Postembryonic development in plants depends on the activity of shoot and root meristems. CLAVATA3 (CLV3), a putative ligand of Arabidopsis thaliana, regulates the stem cell population in the shoot apical meristem (SAM). Previously, we have reported that dodecapeptide with two hydroxy proline residues is a functional CLV3, and chemically synthesized CLV3 dodecapeptide function in our in vitro bioassay systems, resulted in reduced SAM size. In order to confirm the synthetic peptide acts on through endogenous CLV pathway, we examined the SAM phenotype by treating the clv mutants with the CLV3 peptide. As a result, the clv1 and clv2 plants, but not the clv3 plants, showed resistance to the SAM defect caused by the CLV3 peptide. This suggests the synthetic CLV3 peptide acts on through endogenous CLV1/CLV2 receptor complex(es). Together with the analysis of CLV3 peptide-resistant-mutants, we will discuss about CLV signaling pathway in the SAM.

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The plant meristem is maintained by intercellular signalling through dodeca-CLE peptides. The Arabidopsis suppressor of LLP1 2 (sol2) mutant, which suppress the short root phenotype caused by CLAVATA3/ESR-related(CLE)19-overexpression, showed a resistance to CLAVATA3 (CLV3) peptide. We investigated 26 synthetic CLE peptide function in the clv1-4, clv1-6, clv1-13, clv2-1, and sol2 mutants, and suggest that SOL2 and CLV2 function together in CLE signaling pathway in RAM. Positional cloning of the sol2 mutant identified a mutation in a receptor-like kinase protein (At5g13290). Yeast two hybrid screening with kinase domain of SOL2 revealed that SOL2 can interact with kinase, phosphatase and transcription factors. Taken together, we suggest diverse SOL2 functions, including the regulation of shoot and root apical meristem homeostasis through the CLE signaling pathway.

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CLE (CLAVATA/ESR-related) peptides play crucial roles as cell-cell communication factors in plant morphogenesis by regulating cell proliferation and differentiation. We found that TDIF (Tracheary Element Differentiation Inhibitory Factor), which belongs to the CLE family, inhibits differentiation of procambial cells into xylem vessel cells and promotes proliferation of procambial cells. However, it was still unknown if other CLE peptide(s) may function in vascular development. In this report, we searched the function of CLE peptides and found some other CLE peptides than TDIF caused vein discontinuity in leaf margin. TDIF suppressed the vein discontinuity caused to the peptides. Now efforts are underway to reveal the relationship between the CLE peptides and auxin transport.

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Organization and maintenance of the plant meristems are controlled by the balance between cell division and differentiation. Procambium/cambium is the vascular meristem which continuously produces phloem and xylem tissues throughout life. Previously, we reported an intercellular signaling molecule, TDIF, essential for the procambium function. Phloem-derived TDIF peptide is received by an LRR-RLK, TDR/PXY, expressed in the procambium, which leads to the expansion of undifferentiated cell pool. The TDR-mediated TDIF signaling causes two events for the procambium cells: the enhancement of cell division and the suppression of differentiation into xylem cell. In this study, we aimed to identify the target genes in the TDIF signaling. To this end, we analyzed the relationship between TDIF and genes expressed preferentially in the vasculature, and examined transcription factors affecting vascular development. Genetic analysis for candidate genes is also in progress. Taken together, we will discuss the target genes for TDIF.

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The vascular bundle formation in rice leaves occurs one-dimensionally at the base area of the leaf according to developmental program; therefore, we have used rice plants as a model in order to understand the mechanism of vascular bundle formation. Previously, we reported the commissural vein excessive1 (coe1) mutant in which narrower intervals of commissural veins are formed and some commissural veins show the cluster-like structure. In this paper, we revealed that COE1 encodes an LRR-RLK from the complementation test with coe1-1. The intensive analysis of the phenotype of coe1 plants suggested that COE1 is involved in some of brassinosteroid signaling. Based on results obtained so far, we will discuss on the initiation of commissural veins governed by COE1.

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Arabidopsis thaliana VAM3, a syntaxin homolog to yeast Vam3p, is classified as Q_a-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and interacts with AtVTI11 (Q_b-SNARE) and SYP5 (Q_c-SNARE). To elucidate a role of VAM3 in development of plant cells, we generate vam3 of Arabidopsis thaliana. These mutants exhibited the phenotypes of wavy leaves and semi-dwarfism. We focused on vascular cell and myrosin cell development in leaves. Myrosin cells, which play an important role in plant defense, were distributed only along with the veins. vam3 developed a larger number of myrosin cells than wild type. In contrast to the increased myrosin cells, the network of vascular cells was poorly developed in vam3. Interestingly, the network of myrosin cells of vam3 was compensating the immature network of vascular cells. Our findings suggest that VAM3 plays a role in the development of vascular cells and myrosin cells.

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Dof5.8 transcription factor is expressed in provascular cells of leaf primordium in Arabidopsis. Here we show possible involvement of Dof5.8 in vascular development. Based on similar cell-type specificities of the promoters for Dof5.8 and an auxin-responsive marker DR5, we investigated auxin- and the auxin-responsive MONOPTEROS (ARF5) transcription factor-dependent activation of the Dof5.8 promoter in protoplasts. The result revealed that the Dof5.8 promoter and the promoter of OBP1 (Dof3.4), the closest gene of Dof5.8, were similarly activated by auxin and MONOPTEROS. Furthermore, both the provascular-specific expression and the MONOPTEROS-dependent activation of Dof5.8 were shown to require the 5' upstream region of the gene. The ectopic expression of Dof5.8 using the DEX inducible system delayed the xylem differentiation from procambial cells. These results suggest that Dof5.8 is one of the target genes of MONOPTEROS in vascular development in leaves. (This work was supported by PROBRAIN.)

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Plants respond to environmental stresses in multiple ways including changing the hormonal responses. The plant hormone auxin controls every aspect of growth and development. However, little is known about the effect of temperature stress on auxin response. To understand the mechanistic basis of cold temperature stress and auxin response, we characterized the root growth of Arabidopsis thaliana at 23degC after pre-incubating the seedlings at 4degC. The auxin-signaling mutant axr1-3 and tir1-1 responded to cold treatment like wild-type indicating that auxin transport is affected by cold stress. Auxin transport assay further confirmed that cold treatment inhibits root basipetal auxin transport. Additionally, trafficking of auxin efflux carrier PIN2 was dramatically reduced in cold stressed seedlings. Likewise, the endosomal movement was also affected by cold treatment. Taken together these results, we suggest that inhibition of gravity response by cold stress is part of global effect of cold stress on cellular protein trafficking machineries.

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Arabidopsis thaliana acaulis1-1 (acl1-1) phenotype was distinct from its allelic acl1-3 phenotype in the aspect of temperature-dependent restoration of mutant phenotype. Both exhibit growth defects in leaves and inflorescence stems at 22C and those defects are restored and acl1 plants become indistinguishable from wild-type at 28C. However, at 24C, the degree of restoration differs between acl1-1 and acl1-3. Although the acl1-3 plants elongated their inflorescence stems, the acl1-1 plants were similar to those grown at 22C.
We identified an inversion mutation in the original acl1-1 genome (Kamata and Komeda, 2008, Genes & Genetic Systems 83, 293-300). Full-length transcripts of the two genes located at each end of the inversion were not expressed. Expressions of multiple genes within the inversion were also altered. The inversion functioned as an enhancer of the acl1-1 phenotype and thus the elongation of the inflorescence stems of the original acl1-1 plants were inhibited at 24C.

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Roots display tropisms in response to environmental cues such as gravity, light, and moisture by displaying gravitropism, phototropism, and hydrotropism, respectively. Root hydrotropism is considered to have an important role for drought avoidance. However the mechanism underlying root hydrotropism is far from understanding. We previously reported MIZU-KUSSEI1 (MIZ1) as a gene required for hydrotropism, although the function of its gene product was not known. Here, we identified miz2 as a new mutant allele of GNOM, which confers ahydrotropism of Arabidopsis seedling roots. GNOM encodes a guanine-nucleotide exchange factor for ADP-ribosylation factor-type G protein. Unlike other gnom alleles, miz2 showed no apparent morphological defects or reduced gravitropism. On the other hand, brefeldin A treatment, which inhibits GNOM action, inhibited both hydrotropism and gravitropism. These results indicate that a unique GNOM-mediated vesicular trafficking plays an essential role in root hydrotropism.

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The continuous and rapid movement of organelles and endosomal vesicles, which is termed cytoplasmic streaming, is found within plant cells. The cytoplasmic streaming has been studied for two centuries and is believed to be driven by the acto-myosin cytoskeleton. However, the molecular mechanism underlying the cytoplasmic streaming in plants is unknown. In order to elucidate the mechanism and physiological function of the cytoplasmic streaming, we focused on the myosin XI molecules of Arabidopsis thriana which has 13 homologs of myosin XI, and isolated 13 knockout mutants for each gene. However, all of the single mutants did not show any abnormal phenotypes. We further generated double mutants of myosin XI homologs. Finally, we found that one of the double mutants showed a unique phenotype, suggesting that myosin genes have the redundant functions.

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To elucidate the molecular mechanism of gravitropism, we have isolated and analyzed Arabidopsis shoot gravitropism (sgr) mutants. However, further conventional mutant screening mayn't be efficient way to obtain new mutants. Then, we attempted to screen novel genes involved in shoot-gravitropism with DNA microarray analysis comparing gene expression profiles between wild type and several mutants.
Major site for gravity sensing is the endodermis in Arabidopsis shoot. sgr1/scarcrow (scr) and sgr7/short-root (shr) mutant failed to form the endodermis. In addition, our recent study demonstrated that eal1(endodermal-amyloplast less 1), which exhibits no-gravitropism in inflorescence stem but retains ability to form endodermis, is a hypomorphic allele of sgr7/shr.
Take advantage of these mutants, we performed DNA microarray analysis. We found that 29 genes were commonly down-regulated in these mutants. A T-DNA insertion mutant of one gene among them showed reduced shoot gravitropism. Now we are carring out functional analysis of the gene.

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The members of CLE gene family encode 12 amino acid peptides homologous to CLAVATA3 on their C-terminus regions. The CLE peptides are thought to regulate the size of the apical meristem and their differentiation. In this study, we focused on root-expressed NH₄⁺-regulated form, CLE3, of Arabidopsis to elucidate its roles in root development.
Expression of CLE3 was induced by low NO₃^- but was repressed by high NO₃^-. When NH₄⁺ was the sole nitrogen source, CLE3 was repressed by 300 μM NH₄⁺, though increased either by increasing or decreasing the amount of NH₄⁺ in the medium. The CLE3 expression level was negatively correlated with the root fresh weight. CLE3 promoter-GFP plants indicated that CLE3 is expressed in the central cylinder roots. The lengths of the lateral roots were shorter in CLE3 overexpressors than in the wild-type plants, suggesting that CLE3 may act as a repressor of lateral root elongation.

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Programmed cell death(PCD) takes part in an important process of the life system in the higher plant, such as, formation of vascular bundles and hypersensitive cell death against pathogen. Recently, the mechanism of PCD in the plant has been gradually clarified, however, in the case of higher plants, it's difficult to observe the morphological changes in the individual cell during the PCD. So, we paid attention to the using moss plants in which main tissue consists of monolayer cells. When we applied the supernatant of the culture medium of Erwinia carotovora to a model moss plant Physcomitrella patens. 70 % of the chloronema cells died in 24h and their chloroplasts moved to the cell center several hours before the cell death. After the chloroplast aggregation, we succeeded in capturing the vacuolar membrane dynamics during the vacuolar collapse by using a vacuolar membrane marker of AtVAM3-GFP.

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AtLSD1, a negative regulator of programmed cell death (PCD) in Arabidopsis, is a retention protein that inhibits nuclear import of transcription factors (TFs: AtbZIP10, IAA8, CBF-C) by its direct interaction via "GxP" motif. The function of AtLSD1 as a negative regulator of PCD supposed to be derived from that as a retention protein, whereas AtLOL1, AtLSD1 homolog, is known as a positive regulator of PCD. In vivo experiments demonstrated AtLOL1 interacts with same TFs via "GxP" motif as AtLSD1. "GxP" mutation on these TFs, which are localized both in nucleus and cytosol, compromised their cytosol localization. These results suggested that interaction between AtLSD1/AtLOL1 and TFs via "GxP" motif is essential for inhibition of their nuclear import. Since overexpression of AtLSD1ΔC, which lacks C-terminal region of AtLSD1 caused LOL1-like PCD phenotype, this region seems to be involved in antagonistic function of AtLSD1 and AtLOL1 on PCD induction.

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Arabidopsis bZIP transcription factor AtbZIP10 involved in programmed cell death (PCD) specifically interacts with microtubule plus-end tracking protein AtEB1a and AtEB1b. If AtEB1 regulates AtbZIP10's nucleocytoplasmic shuttling, AtEB1s would have an important function to induce PCD. Here, we investigated function of AtEB1 on PCD inducing mechanism as well as details of protein-protein interaction. Co-expression of AtEB1 slightly enhanced signal derived from AtbZIP10-GFP, which was transiently expressed in Arabidopsis mesophyll protoplasts, in cytoplasm. In addition, this enhanced signal of AtbZIP10-GFP in cytoplasm was dramatically amplified by treatment of microtubule depolymerizing agent Taxol. These results suggested specific interaction between AtEB1a,b and AtbZIP10 at plus-end of microtubule participates in AtbZIP10's nucleocytoplasmic shuttling. Since Fumonisin B1 induced higher PCD level in AtEB1a overexpressor, AtEB1's function may be involved in PCD inducing mechanism.

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AAL-toxin is a pathogenicity factor of Alternaria alternata f. sp. Lycopersici, a pathogen of tomato stem canker disease. Little is known about the signaling pathway of AAL-toxin leading to programmed cell death. To dissect the biological function of genes involved in AAL-toxin-induced cell death, we used Nicotiana umbratica that is sensitive to the AAL-toxin, and virus-induced gene silencing (VIGS). We have showed that ethylene signaling plays a pivotal role in AAL-toxin-induced cell death and identified NuERF4 as a component for the cell death signaling. Here, we investigated regulatory mechanism of NuERF4 for transcription using GAL4 cis-element and minimal 35S promoter, indicating that NuERF4 positively activated the transcription. Overexpression of NuERF4 did not induce cell death in N. umbratica, however, accelerated AAL-toxin-induced cell death and disease symptom by the pathogen inoculation. These results suggest that NuERF4 positively regulates genes involved in AAL-toxin cell death.

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Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. It is no doubt that autophagy, a bulk protein degradation system, is very important in various aspects in plant life.
So far, we have clearly indicated that ATG (autophagy-related) genes-disrupted plants (atg mutants) are defective in autophagy, and the mutants exhibited early senescence phenotype. However, relationship between the phenotype and roles of autophagy has not been explained yet.
Recently, we found that the early senescence phenotype in the atg mutants was clearly suppressed by over expression of NahG gene which encodes a bacterial hydroxylase that converts SA to catechol, thereby reducing SA accumulation. In addition, the suppression was canceled by BTH which is a functional analog of SA, suggesting that SA signaling pathway is activated in the autophagy-defective mutants after SA biosynthesis. Autophagy may be involved in the elimination of SA signaling.

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The circadian clock is involved in the regulation of various physiological phenomena. In Arabidopsis, two closely related MYB proteins LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) are essential clock components with redundant functions. Arabidopsis plants with mutations in LHY and CCA1 genes exhibited not only late flowering, but also semi-dwarf phenotypes under continuous light (LL). We have recently found that the late flowering phenotype of lhy;cca1 was suppressed by mutation of elf3, svp and flc (Fujiwara et al., Plant Cell 2008). To understand molecular mechanisms underlying these phenotypes, we have isolated 7 enhancers (petanko 1-7; pta1-7) and 1 suppressors of the phenotypes of lhy;cca1 under LL. We will show results on pta1 and discuss possible roles of PTA1 in the control of flowering time and organ elongation with LHY/CCA1.

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Fine-tuning of morphology and length of leaf blade and petiole is quite important for efficient photosynthesis of plants under a variety of environments they live. Plant hormones and light signaling play key roles in the control of the shape of leaves. We have found that circadian clock also has some effects on the control of leaf shape and length. Mutations in two clock genes LHY and CCA1 (lhy;cca1) change flowering time and lengths of hypocotyls and petioles. Under continuous light (LL), the lhy;cca1 plants have late flowering and semi-dwarf phenotypes (Fujiwara et al., Plant Cell 2008). To understand molecular mechanisms underlying these phenotypes, we have isolated 7 enhancers (petanko 1-7; pta1-7) and 4 suppressors of the phenotypes of lhy;cca1 under LL. The pta5 was mapped to the lower side of Ch3. We will discuss possible roles of PTA5 in the control of flowering time and organ elongation with LHY/CCA1.

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EARLY FLOWERING 3 (ELF3) plays key roles in the control of plant morphology, flowering time and circadian rhythms. However, it has not been elucidated how ELF3 protein accomplishes the multi-tasks. Also there has been less information on direct interactors for ELF3 and mutations that suppress the phenotypes of elf3.
In order to isolate suppressors and enhancers of elf3-1, heavy ion beams mutagenesis was performed using the elf3-1 seeds and M2 populations were screened under continuous light condition based on 3 phenotypes: flowering time, hypocotyl length and leaf size. According to these criteria, 7 suppressors and one enhancer of elf3-1 have been identified. These suppressor mutations were named suppressor of elf3 1, 3, 5, 7, 14, 15 and 20 (sel1, 3, 5, 7, 14, 15, and 20). We will discuss possible roles of these suppressor and enhancer genes in the multiple functions of ELF3.

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EARLY FLOWERING 3 (ELF3) plays key roles in the control of light response, organ elongation, flowering time and circadian rhythms in Arabidopsis. ELF3 does not show significant homology to any other proteins in the public databases. Therefore biochemical function of the ELF3 has not been well understood.
To understand molecular mechanisms underlying the variety of phenotypes in the elf3 mutants, suppressors of elf3-1 were screened , using the elf3-1 seeds mutagenized with heavy ion beams. Here we demonstrate that early flowering, small cotyledon and pale green phenotypes of elf3-1 were suppressed in one of the suppressor lines, S#20, whereas long hypocotyl phenotype was enhanced. The recessive suppressor mutation was named suppressor of elf3 20 (sel20) and mapped to the upper side of Ch1. We have found 21-bp deletion in the FHA/CRY2 gene in the sel20. We will discuss possible roles of CRY2 in the multiple functions of ELF3.

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Tomato is an excellent model plant for studying development, ripening and metabolism of fruits. The International Solanaceae Project (SOL, www.sgn.cornell.edu) was initiated by members from more than 30 countries in 2003, and the tomato genome projects are currently underway.
As a contribution of the Japanese SOL (JSOL) to the international collaboration, we started generating T-DNA tagged lines using a miniature tomato cultivar Micro-Tom. T-DNA insertional mutagenesis is a complementary approach to that by EMS and provides a rapid way to isolate mutated genes. More than 80 independent lines transformed with T-DNAs were generated. Southern blot analysis indicated that average copy number of T-DNA per the transgenic plant was 1.86. Of 109 T-DNA flanking sequences obtained, 60 sequences correspond to those from the Micro-Tom genome. We will show a current status of this project including some visible phenotypes of the T-DNA tagged lines and map-positions of the T-DNA insertions.

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In higher plants, circadian clock controls various biological processes including organ growth. Mutations of circadian clock components often alter organ size, but the mechanism on the clock-controlled regulation of organ size has remained unclear.
We have focused on the analysis of the Arabidopsis lhy;cca1 mutants that have a semi-dwarf phenotype under continuous light (LL). We found that the small leaf area observed in LL-grown lhy;cca1 plants is probably caused by reduced cell growth.To understand roles of circadian clock in the control of organ size, we analyzed the ploidy levels in clock mutants including lhy;cca1. Furthermore, we screened for suppressors of the dwarf phenotype of lhy;cca1. The mechanisms that underlie the dwarf phenotype in lhy;cca1 will be discussed in more detail.

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Genes in the FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) family have been shown to be important in the control of the switch between vegetative and reproductive growth in several plant species. We have isolated nine member of the FT/TFL1 family from Lombardy poplar (Populus nigra L. var. italica). We determined the PnTFL1 is a functional gene in the TFL1 clade of Lombardy poplar by the analyses of its expression levels and pattern, and by the phenotypes of transgenic Arabidopsis over-expressing this gene. Then, we produced PnTFL1-RNAi transgenic poplar, and these transgenic poplars showed early flowering phenotypes. The PnFT1 and the PnFT2 is functional FT gene in Lombardy poplar by the similar analyses of the TFL1 clade. On the other hand, the transgenic poplar that over-expressing the PnFT1 or the PnFT2 never flowered by higher expression of those gene.

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Photoperiods control several morphogenic responses such like flowering throughout plant life cycle. Recently, Hd3a was shown to be an important component of the mobile florigen signal. To study the function and localization of Hd3a in rice, we expressed the Hd3a-GFP gene by fusing it with phloem specific Hd3a promoter. Transgenic rice plants flowered significantly earlier than wild type, suggesting that expression of Hd3a-GFP causes early flowering. To examine tissue localization of the Hd3a in Hd3a-GFP transgenic plants, we analyzed GFP fluorescence in the SAM by confocal laser scanning microscopy. The GFP signal was detected in the SAM. There are some mechanisms that regulate the movement of Hd3a from the vascular bundles into the SAM. But there mechanisms are unclear. So, we study how Hd3a is translocated from the vascular bundles into the SAM and Hd3a regulated in the SAM.

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Heading date 3a (Hd3a) protein in rice and its Arabidopsis ortholog FT are recently considered as the likely florigen, the crucial component of a long-distance flowering signal. Hd3a protein is generated at the leaf phloem tissue in flowering-promoting conditions, moves from leaves to apical meristem and causes flowering. Here we show overexpression of Hd3a in vasculature caused not only early heading but also high tillering, semi-dwarf, abnormal internode elongation and abnormal panicle architecture. Detailed observation revealed that high tillering phenotype is caused by outgrowth of axillary meristems. We show that Hd3a protein moves from leaf to the axillary meristems and induces meristem outgrowth. These data suggest that Hd3a/FT-like proteins may define the novel type of the mobile molecule that integrates the development between organs that sense and adapt the environment.

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Hd3a and FT proteins have been recently proposed to act as florigens in rice and Arabidopsis, respectively. However, molecular mechanisms of their function remain to be determined. In this study, we identified GF14c (14-3-3 isoform protein) as an Hd3a interacting protein in yeast two-hybrid screen. In vitro and in vivo experiments, using a combination of pull-down assay, co-immunoprecipitation and bimolecular fluorescence complementation, confirmed the interaction between Hd3a and GF14c.
Functional analysis using either GF14c overexpression or knockout transgenic rice plants indicated that GF14c plays a role in regulation of flowering. GF14c-overexpressing plants exhibited a delay in flowering and the knockout mutants displayed early flowering that was comparable to wild-type plants under short-day conditions. Taken together, these results suggest that GF14c acts as a negative regulator of flowering by interacting with Hd3a.

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RICE FLOWERING LOCUS T 1 (RFT1) is the closest homolog to Heading date 3a (Hd3a), which promotes floral transition in short-day (SD) in rice. We demonstrate that RFT1 is a floral activator under long-day (LD) conditions. An RFT1:GFP fusion protein localized in the shoot apical meristem (SAM) under LD conditions, suggesting that RFT1 is a LD-specific florigen gene in rice. Furthermore, mutants in OsMADS50, a rice ortholog of Arabidopsis SUPPRESOR OF OVEREXPRESION OF CONSTANS 1 (SOC1) did not flower up to 300 days after sowing under LD conditions, suggesting that OsMADS50 is also a positive regulator of flowering in response to LD and the OsMADS50-Ehd1-RFT1-OsMADS14 and OsMADS15 pathway is involved in floral activation under LD conditions. We propose that both positive (OsMADS50, Ehd1 and RID1/Ehd2/OsId1) and negative (Hd1, phyB and Gdh7) regulators of RFT1 form a gene network which regulates LD flowering in rice.

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Rice is a facultative short day plant, and molecular genetic studies have identified the major genes involved in short day flowering. We focused on study of the natural variation of photoperiodic flowering pathway in cultivated rice. The aim of this study is to understand the mechanism which generates the diversity of flowering time in cultivated rice.
In this study, a high correlation was observed between the levels of Hd3a expression and flowering time. Additionally, high degree of nucleotide polymorphism affecting its protein function was found in Hd1, and cultivars with non-functional Hd1 allele showed relatively low Hd3a expression and late flowering phenotype. Together, these results suggest that the diversity of Hd1 is one of the main sources for generating diversification of flowering time in cultivated rice. Furthermore, diversity in Hd3a promoters and Ehd1 expression level is closely associated with the diversity in Hd3a expression level.

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In 1943, Chl d was first reported as a minor pigment in several red macroalgae. A novel cyanobacterium, Acaryochloris marina, was isolated from colonial ascidians, and A. marina was found to contain Chl d as the dominant chlorophyll. However, the biosynthesis pathway of Chl d has not been clarified yet. Chl d is thought to be synthesized from Chl a, like Chl b, while no experimental evidence is present. We came across the formation of Chl d from Chl a in organic solvents containing 10 % water in the presence of papain (EC 3.4.22.2) at room temperature in the dark; Chl d was not formed when papain was absent. The conversion of Chl a into Chl d in aqueous acetone in the presence of papain significantly increased, when incubated at 70^oC.

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The chlorin ring structure is formed by the reduction of the porphyrin D-ring by a nitrogenase-like enzyme, dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) in the biosynthetic pathway of bacteriochlorophyll. In the previous work, we proposed a reaction mechanism for the stereo-specific reduction of Pchlide D-ring (C17-C18 double bond) based on the X-ray crystallographic structure of the DPOR catalytic component NB-protein (BchN-BchB heterotetramer) from Rhodobacter capsulatus. In this study, we isolated and characterized a series of NB-protein mutants in which some amino acid residues expected to be involved in the D-ring reduction were altered. In addition, we also examined the effect of a substrate analogue chlorophyll c, which has an acrylate at the C17 position instead of the propionate, on DPOR reaction. The results were consistent with the involvement of BchB-Asp274 and the propionate at C17 of Pchlide as the proton donors for C17 and C18 carbons, respectively.

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Dark-operative protochrolophyllide reductase (DPOR) is a nitrogenase-like enzyme consisting of three subunits, ChlL, ChlN and ChlB, and plays a critical role in chlorophyll biosynthesis in the dark. DPOR is distributed widely not only in anoxygenic photosynthetic bacteria but also in oxygenic phototrophs, which raises a question how the nitrogenase-like DPOR operates in oxygenic photosynthetic cells. Here we report functional expression of cyanobacterial DPOR compornents in the cyanobacterium Leptolyngbya boryana to examine the oxygen sensitivity. We constructed plasmids for overexpression of ChlL and ChlN-ChlB. We detected unambiguous DPOR activity by mixing two crude extracts of L. boryana overexpressing ChlL and ChlN-ChlB, allowing to evaluating the activities of L-protein and NB-protein individually. L-protein activity in the extract was almost lost upon the exposure to oxygen for 10 min, while NB-protein activity was maintained after 60 min exposure. These results suggest that L-protein is the target of oxygen inactivation in cyanobacterial cells.

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Chlorophyll b reductase catalyzes the conversion of chlorophyll b to 7-hydroxymethyl chlorophyll a, which is the first step of chlorophyll degradation pathway. There are two isoforms of chlorophyll b reductase, NYC1 and NOL, in Arabidopsis. We analyzed various nyc1 and nol mutants to understand the physiological functions of chlorophyll b reductase. Chlorophyll a/b ratio increased in nol overexpression mutant in green leaves. nyc1 knockout mutant showed the phenotype keeping green color during senescence due to the inhibition of chlorophyll degradation. On the other hand, nol mutant did not show stay-green phenotype. Based on these observations, we will discuss the functions of chlorophyll b reductase in the developmental stages of seed formation, germination, greening and senescence in Arabidopsis.

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During leaf senescence, most of chlorophyll is degraded which results in yellowing, the unmasking process of the preexisting carotenoids in leaves. Several enzyme genes involved in the chlorophyll breakdown pathway have been isolated through biochemical and genetic approaches. nyc1 and nol are also non-functional stay green mutants. Their phenotypes are very similar because NYC1 and NOL form the Chl b reductase complex catalyzing the first step of Chl b degradation.Here we report a new non-functional stay-green mutant in rice, nyc3. nyc3 shows distinct phenotype from nyc1 and nol. Map-based cloning revealed that NYC3 encodes a plastid-localizing α β hydrolase-fold family protein. The possible function of NYC3 in the regulation of chlorophyll degradation is discussed.

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Coproporphyrinogen III oxidase (CPO) catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX in heme biosynthesis that is shared with chlorophyll biosynthesis in photosynthetic organisms. There are two structurally unrelated CPOs; oxygen-dependent (HemF) and oxygen-independent (HemN) oxidases. The cyanobacterium Synechocystis sp. PCC 6803 has one hemF-like gene, sll1185, and two hemN-like genes, sll1876 and sll1917. Here we report the biochemical analysis of these CPO-like proteins. Purified Sll1185 showed CPO activity in aerobic condition, indicating that sll1185 encodes oxygen-dependent CPO, HemF. While both purified Sll1876 and Sll1917 showed absorption spectra characteristic of a [4Fe-4S] cluster, only Sll1876 showed CPO activity dependent on NADH, indicating that sll1876 encodes oxygen-independent CPO, HemN. Given the previous phenotypic analysis of the mutants, it was suggested that HemF and HemN operate as the dominant CPOs under aerobic and micro-oxic conditions, respectively, in the heme biosynthetic pathway of Synechocystis sp. PCC 6803.

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Prasinophyceae are primitive eukaryotic green algae. These ubiquitous marine picophytoplankton contribute considerably to global carbon-fixation. Prasinophytes contain unique antenna proteins, dubbed Lhcp, which predate modern light-harvesting complex (LHC). Ostreococcus tauri, the first prasinophyte to be completely sequenced, is an ideal species for primitive algal studies. Characterization of photosynthetic systems in this species will clarify early stages in eukaryotic photosynthetic evolution. In this study we isolated O. tauri LHC, photosystem I, and photosystem II complexes using sucrose density gradient centrifugation. This reveals novel PSI complexes formed by strong association with both Lhca (PSI-specific LHC) and Lhcp. Both this complex and purified PSI-core are devoid of the far-red chlorophyll fluorescence (>700 nm) typical of PSI in other oxygenic phototrophs. Here we discuss the implication of this PSI-Lhcp association and the full characterization of O. tauri photosynthetic components using a variety of biochemical techniques, including HPLC, spectroscopy, and proteomic identification by mass spectrometry.

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The stacking of the bacteriochlorophyll (BChl) c and the role of water in forming an aggregate, in chlorosome, were examined by ¹³C-NMR and ²⁵Mg-NMR spectroscopies and X-ray diffraction: (1) "The weakly-overlapped dimers forming displaced layers" was selected out of six different stackings so far identified in the smaller aggregates of isomeric BChl c and the solid aggregate of an isomeric mixture of BChl c. The selection was based on the comparison between the intermolecular ¹³C magnetic-dipole correlations and the nearest-neighbor carbon-to-carbon close contacts. (2) The crucial role of water in stabilizing the aggregate was shown by tracing the dehydration processes and by comparison with the solid-aggregate X-ray diffraction pattern. Possible binding sites of water were located by structural simulation. (3) The dimer-based stacking was evidenced by ²⁵Mg-NMR, which exhibited a pair of signals showing different quadrupole couplings, due to the presence or absence of water in the axial position.

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