Cytokinin inhibits root formation and activates shoot formation in tissue culture. This suggests that cyokinins regulate processes that govern root/shoot decision. We analyzed 16 genes that were strongly repressed by cytokinins in the cytokinin hypersensitive mutants, ckh1, and ckh2. We first examined expression patterns of these genes. 6 genes were specifically expressed in the root apex. To understand the functions of these genes, we overexpressed these genes, and found a gene that induced roots on cotyledons when overexpressed. This gene codes for an uncharacterized transcription factor. This gene, when fused to the sequence for SRDX repressor domain, gave shoot properties to the root. These results suggest that this gene potentially confers root identity to stem cells, and prevents the root stem cells from expressing shoot properties.

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Legume plants have developed an intimate association with nitrogen-fixing rhizobial bacteria that provide plants with a reliable nitrogen source. Central to this plant-bacterial interaction is the formation of unique organs on plant roots called nodule. The nodules are plant-derived structures that formed from cortical cells following recognition of rhizobial bacteria by the plant. The phytohormone cytokinin is a key element in the nodule formation. Several reports of gain-of-function mutation and loss-of-function mutation in Lotus japonicus Lhk1 that is a homologous gene of Arabidopsis CRE1 encoding the cytokinin receptor revealed that Lhk1 is required for the initiation of nodule development. Although, the molecular mechanisms that cytokinin signaling controls the nodule development remain largely unknown. The mechanism of cytokinin signaling and some factors involved in the pathway have been revealed in Arabidopsis.
In this presentation, the involvement of the homologous genes in the cytokinin signaling in the nodulation of Lotus japonicus root, will be reported.

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Cladosiphon okamuranus is an original strain of a brown alga in Okinawa, which have fucoxanthin-chlorophyll a/c proteins (FCP) as a major photosynthetic antenna complexes. FCP is thought to transfer energy to the oxygenic photosystem I and II, although its detail is not clarified yet. Recently Iha et al. has been successful to culture a discoid germiling of a brown alga, which is a precursor of an alga body. We established the isolation and purification method of the FCP from the discoidal form. We also found the FCP exists as a homo- or hetero-trimer formed by the products of three highly homologous subunits in unequal stoichiometries. Hereafter, we call this particular complex as 'mozuku FCP'. In this study, we determined the optical-properties of the pigments binding in the 'mozuku FCP', which has high biochemical degree of purity. First, we determined the pigment composition of the 'mozuku FCP' using HPLC and ¹H-NMR. Then, we also measured the fluorescence and fluorescence-excitation spectra of the 'mozuku FCP' to determine the efficiencies of energy transfer from both fucoxanthin and Chl c to Chl a.

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To clarify the mechanism of the coordinate gene expression for assembly of photosynthetic apparatus, we studied plastic chloroplast differentiation in Arabidopsis roots. For root greening, we have clarified that light signaling mediated via HY5 is indispensable, and cytokinin and auxin signals oppositely regulate the coordinate expression for the root greening, which are mediated by other transcription factors. Recently, GARP transcription factor, Golden2-like (GLK), is identified to control genes involved in photosynthetic apparatus. Expression analysis of Arabidopsis isoforms, GLK1 and GLK2, in greening roots revealed the induction of GLK2, while GLK1 mRNA remained undetectable. Chlorophyll accumulation in double mutant glk1 glk2 was significantly lower than WT, while those in overexpressors GLK1_OE and GLK2_OE were approximately 6 and 3 fold higher than WT, respectively. These results suggest that the phytohormonal control of chloroplast differentiation is primarily mediated via GLK2 in Arabidopsis roots. As exogenous treatments of cytokinin or anti-auxin PCIB still enhanced chlorophyll accumulation in these lines, other factors may also be involved in this regulation.

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The dark-grown angiosperms cannot convert protochlorophyllide (PChlide) to chlorophyll (Chl) and are etiolated in time. In etiolated chloroplasts and etioplasts, PChlide is localized in prothylakoids (PT) and prolamellar bodies (PLB), giving fluorescence peaks at 633 and 657 nm, respectively. Upon the illumination of etioplasts, PChlide is converted into Chl quickly and assembly of photosystem starts. We performed confocal microspectroscopy at 90 K to stop greening process, to clearly identify PChlide and Chl on different complexes and to study assembly of photosystems during the greening process.
We used a C₄ plant Zea mays. C₄ plants show different distribution of photosystem I and II between bundle sheath and mesophyll cells. It is interesting how the differentiation of pigment systems in these cells proceeds during greening. We found the ratio of PLB/PT fluorescence to be different between cells near vessel bundle and the outer surrounding cells in etiolated leaves. Two fluorescence bands at about 680 nm and 720 nm emerged after 3 h illumination, and the photosystem I 720 nm fluorescence became higher in cells around vessel bundle, suggesting the differentiation process.

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Dark-operative protochlorophyllide(Pchlide) oxidoreductase (DPOR) catalyzes the stereo-specific reduction of D-ring double-bond of protochlorophyllide to form chlorophyllide a. DPOR consists of two separable components, L-protein (BchL homodimer) and NB-protein (BchN-BchB heterotetramer), which are homologous to Fe protein and MoFe protein of nitrogenase, respectively. In our previous work, we have solved the crystal structure of NB-protein from Rhodobacter capsulatus and proposed a catalytic mechanism of Pchlide reduction. However, 110 amino acids residues in C-terminal region of BchB were disordered. This region is conserved in BchB(ChlB) but not in NifK of nitroganase MoFe-protein, suggesting that the region has unique role(s) in DPOR reaction. To elucidate the functional significance of the C-terminal region, we have constructed a series of BchB mutants in which the C-terminal region was deleted partially or completely. SDS-PAGE profiles of the purified truncated BchB showed doublets with BchN, indicating that the C-terminal region is not essential for BchN-BchB complex formation. Characterization of the mutant NB-proteins is in progress.

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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 (Chl) biosynthesis in the dark. While nitrogenase is distributed only among some prokaryotes, there has been no direct evidence for functional operation of nitrogenase-like enzyme in any eukaryotes. Here we report functional expression of probable DPOR components encoded by the chloroplast genome of Physcomitrella patens in the cyanobacterium Leptolyngbya boryana. We constructed two shuttle vectors for overexpression of two DPOR components, ChlL and ChlN-ChlB from P. patens, and introduced them into the cyanobacterial mutants lacking chlL and chlB. Both transformants restored the ability of Chl biosynthesis in the dark, indicating that these chloroplast genes encode functional DPOR subunits and that each DPOR component is compatible between P. patens and L. boryana. This compatibility was also confirmed by in vitro reconstitution with purified ChlL and ChlN-ChlB.

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Heme oxygenase (HO) catalyzes the oxygen-dependent cleavage of porphyrin ring of heme, producing biliverdin IXα in phycobilin biosynthesis. In the genome of the cyanobacterium Synechocystis sp. PCC 6803 there are two genes, ho1 (sll1184) and ho2 (sll1875), encoding heme oxygenase isoforms. We have isolated two mutants, Δho1 and Δho2, in which ho1 and ho2 were inactivated, respectively. The Δho1 mutant failed to grow under aerobic conditions and grew significantly slower than wild type under anaerobic (micro-oxic) conditions. The Δho2 mutant grew as well as wild type under both conditions. This result suggested that ho1 is essential for growth under aerobic conditions and is dispensable under micro-oxic conditions and that HO1 operates together with HO2 induced by oxygen-limited conditions. To understand the physiological significance of HO2, we are examining whether overexpression of ho2 restores the growth defect of Δho1. We will also present pigment analysis of these mutants.

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Phytol is a diterpene alcohol and a precursor for the biosynthesis of tocopherol, chlorophyll and phylloquinone. In plants, phytol is synthesized through the reduction of another diterpne, geranylgeraniol. A soluble chloroplast protein, geranylgeranyl reductase has been identified to catalyze the reduction of geranylgenraniol to form phytol. This protein is encoded by a nuclear gene, ChlP. We have previously shown that the suppression of ChlP expression results in the reduction of the tocopherol levels and in the occurrence of chlorophyll derivatives conjugated with a partially reduced geranylgeranyl side chain. In the course of screening for Arabidopsis mutants that were defective in chlorophyll metabolism, we isolated two mutant lines showing very similar phenotypes to those of the ChlP suppression line mentioned above. Therefore, we hypothesized that the activity of geranylgeranyl reductase was reduced in these mutants. We identified the mutations in a pair of homologous genes encoding chloroplast membrane proteins with unknown functions. These results indicate that chloroplast membrane-proteins other than geranylgeranyl reductase are involved in phytol biosynthesis.

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Photosynthetic organisms have evolved to synthesize various photosynthetic pigments. It would be reasonable to assume that the modification of pigment-binding proteins was an essential step for the acquisition and utilization of new photosynthetic pigments. For better understanding of such adaptation processes of pigment-binding proteins, we attempted to reproduce them in a laboratory.
We first disrupted the gene essential for monovinyl-chlorophyll (MV-Chl) synthesis in the Synechocystis sp. PCC6803, and isolated a mutant accumulating divinyl-chlorophyll (DV-Chl), which pigment is only found in nature in a few groups of cyanobacteria. Accumulation of DV-Chl in this mutant made it highly susceptible to strong illumination. Subsequently, we introduced a base substitution to the genes encoding a chlorophyll-binding protein, D1, so that the mutated D1 sequences obtained the amino acids that are only conserved in the DV-Chl producing cyanobacterial species. In a consequence, the Synechocystis mutant became less sensitive to illumination. These results indicate that modification of chlorophyll proteins is critical to utilize a newly acquired pigment.

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Plants produce two chlorophyll species, chlorophyll a and chlorophyll b. These chlorophylls are enzymatically converted to each other within the chloroplast. The conversion of chlorophyll b to chlorophyll a is a two-step reaction, the first of which is catalyzed by chlorophyll b reductase. We have previously shown that this enzyme is essential in the breakdown of chlorophyll b, LHCII and thylakoid membranes during leaf senescence. Since the gene for this enzyme is highly expressed during seed maturation as well, we speculated that this enzyme also functions in maturing seeds. In this study, we investigated a role of this enzyme in seed maturation by analyzing an Arabidopsis mutant that lacks chlorophyll b reductase. We found that a large amount of chlorophyll remained in the mature seeds in this mutant. The germination rate of the mutant seeds decreased with increasing storage periods. The cotyledons of the mutants often variegated or went bleached, which is most likely due to a photodamage by excessive chlorophyll. These results suggest that chlorophyll b reductase plays an important role in the seed maturation process.

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Chlorophyll is a source of reactive oxygen species in plants; thus it must be converted to safe molecules. Identification of the enzymes of the chlorophyll degradation pathway suggest that many steps in this pathway require reducing power. However, what mechanisms supply reducing power to chlorophyll degradation is not well understood. To understand these mechanisms, we isolated a chlorophyll degradation mutant,hmc2, which accumulates two intermediate molecules. It would be reasonable to assume that accumulation of these compounds were due to a reduction in the activity of both 7-hydroxymethyl chlorophyll a reductase and pheophorbide a oxygenase, which catalyze the catabolism of 7-hydroxymethyl chlorophyll a and pheophorbide a, respectively. Interestingly, both of these enzymes require reducing power for their reactions. Since the HMC2 gene encodes a flavin binding motif, we hypothesized that HMC2 protein supply reducing power to these reactions. The spectral analysis of the recombinant protein exhibited the absorbance peak at 460 nm which corresponds to flavin. We will discuss a possible role of HMC2 in the distribution of reducing power to the chlorophyll degradation pathway.

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Free chlorophylls should be degraded immediately to detoxify their phototoxicity. In the chlorophyll degradation pathway, RCCR catalyzes the reduction of the C20/C1 double bond of RCC to produce pFCC. Recently we determined the crystal structure of Arabidopsis thaliana RCCR, in which RCCR subunit folds in a characteristic α/β/α sandwich observed in FDBR family.
Here we have determined the crystal structures of RCC-bound RCCR and its F218V mutant, by which the stereoisomer of pFCC at the C1 position is produced. These structures demonstrate that RCC is bound on the pocket between the β-sheet and the C-terminal α-helices of RCCR. The substrate binding mode is similar to that of FDBR, but the substrate appears to loosely bind on RCCR. It is reasonable because the reaction intermediate of RCCR is estimated to be more bulky than RCC. Glu-154 and Asp-291 are nearby the reduction site of RCC, suggesting that these residues function as acid catalysts. Structural comparison between wild-type RCCR and F218V mutant suggests that the catalyst attacking the RCC C1 position is different between them, reflecting the chirality of pFCC produced.

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The biosynthetic pathway of Chl d in Acaryochloris marina is not clear. Chl d-derivative was first synthesized artificially from Chl a-derivative by Mironov group in 1994, but the reaction requires strong oxidant (e.g., H₅IO₆) and special metal complex (e.g., OsO₄), and highly unlikely to occur in nature. Recently, we came across the formation of Chl d from Chl a catalyzed by papain in aqueous organic solvents at room temperature. Papain is a proteolytic and thiol protease with a relatively low selectivity. The novel conversion was not observed when Chl a was incubated with esterases (esterase, cholesterol esterase, phosphatase) and other proteases (α-chymotrypsin, bromelain, ficin, subtilisin carlsberg). In order to clarify the conversion mechanism in vitro and the origin of conversion of Chl a into d in nature, we incubated Chl a with extracts of several vegetables and fruits. The conversion occurred when papaya(skin), cucumber(skin), scallion(leaf), white radish(leaf) and Japanese radish(leaf) were used. Chl d was not present in their initial skin, leaf and bean. The present results suggest that the conversion of Chl a intod is not a rare event in nature

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Until 1993, Chl a had been believed to be essential in oxygenic photosynthesis without exception. However, Chl d is found to be dominant in a unique cyanobacterium Acaryochloris marina. From the molecular structure of Chl d, Chl d is expected to be oxidatively biosynthesized from Chl a, like Chl b. However, the biosynthetic pathway of Chl d in A. marina is not clear. The conversion of Chl a to Chl d requires an oxidative cleavage of the C=C double bond of a vinyl group of Chl a at ring I (-CH=CH2 into -CHO). We serendipitously came across the formation of Chl d from Chl a by papain in organic solvents containing water. Recently, we found that the novel conversion happened when Chl a was incubated with extracts of several vegetables. Here, we will report the Chl a to Chl d conversion catalyzed by extracts of several microalgae in aqueous acetone. The findings suggest that the conversion of Chl a into Chl d is one of the degradation reactions of Chl a in vitro, like pheophytinization and epimerization, and that the conversion may not be a rare event in nature. Our findings will provide new insight into the unsolved question as to the birth of Chl d.

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Acaryochloris spp. contains chlorophyll (Chl) d as a major Chl and also a small amount of Chl a, and can utilize infrared light for photosynthesis. Recently, the complete genome of the type strain, Acaryochloris marina MBIC 11017, was sequenced, however, lack of a genetic technique prevented us from studying A. marina intensively by a molecular genetic approach. In this study , we developed the technique for gene transfer into A. marina, and succeeded in producing a transformant expressing a foreign gene. First, we produced an expression vector from a broad-host range plasmid, and tried to introduce the vector into A. marina by conjugal gene transfer. Colonies obtained by antibiotic selection retained the vector. Then, we produced a transformant expressing chlorophyllide a oxygenase gene (CAO) that involved in Chl b biosynthesis. The pigment composition of the transformant was analyzed by HPLC, and the accumulation of a novel pigment was found. Based on the absorption spectrum, we estimated it to be a Chl derivative. The chemical structure, properties and proposed biosynthetic pathway of the novel Chl will be discussed.

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Since the discovery of Acaryochloris marina by Miyashita et al[1], many studies have revealed that this organism with Chl d can do oxygen-evolving photosynthesis as efficiently as other cyanobacteria with Chl a do. It is now known that PS I RC of this organism has Chl d/ Chl d'heteromer as the electron donor special pair P740(2), and PS II has Chl d/Chl ddimer (3-5) or Chl d/Chl a heteromer[6] as the special pair.It is surprising how the exchange of Chl a to Chl dsmoothly occurred without damaging photosynthetic systems. However, we also know that photosystems in PS I and II are highly optimized so that only a mutation of one amino acid sometimes destroy the whole photosystem.We feel that exchange of Chl has opened a big chance to this cyanobacteria to change its system still keeping high efficiency and allowed diverse change of the system as animals in Galapagos experienced."Why ?"We will discuss based on some unpublished results.1.Miyashita, H., et al. (1996). 2. Hu, Q., et al. (1998). 3. Itoh, S., et. al (2002). 4. Itoh, S. et al (2007) 5. Tomo T. et al (2007) 6. Schlodder, E. et al.(2007)

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Plant cells have numerous single-membrane organelles such as the Golgi body, vacuole, and endosome. Components such as cargo proteins and lipids are transported between these single-membrane organelles and the plasma membrane in membrane trafficking. Recently, live imaging techniques using fluorescent tags and fluorescent tracers have enabled the detection of the organelles and vesicles. However, ultrastructures such as Golgi vesicle and endosome are not clearly detectable in the plant cell. Therefore, we are performing an exhaustive ultrastructural analysis of single-membrane organelles in Arabidopsis and tobacco roots using high-pressure freezing and electron microscopic techniques. We observed highly dense large vesicles derived from the Golgi body in epidermal cells but not in columella cells. Meanwhile, many clusters of vesicles were existed in columella cells but not in epidermal cells. Moreover, the number and structure of the multi-vesicular body and Golgi body varied in columella cell layers. Here, we will mainly discuss columella cells.

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Phosphatidylinositol 3-kinase (PI3K) produces phosphatidylinositol 3-phosphate, a signaling lipid required for various biological pathways, such as autophagy and endocytosis. In plants, the roles of PI3K complexes in cellular activities remain obscure, because no pi3k mutant is viable to date. We previously showed that each of AtAtg6, AtVps34 and AtVps15 in the Arabidopsis PI3K complexes is required for pollen germination. In this study, we created atatg6 homozygous plants whose defects in pollen germination were rescued by pollen-specific overexpression of AtAtg6. The resultant atatg6 mutant not only showed defects in autophagy but also exhibited severe growth defects. This mutant should serve as a model system for further investigating the physiological functions of plant PI3K complexes.

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Directional movement of motor protein is essential for regulation of intracellular trafficking. In animal cells, microtubule and motor proteins play important roles on trafficking. In contrast, in plant cells, the actomyosin system takes the place. Although actin related motor is only plant specific class VIII and XI myosins, higher plant Arabidopsis thaliana developed many members, 4 in myosin VIII and 13 in myosin XI. Our goal is an integrated understanding of the unique plant trafficking regulated by myosin members.
I have cloned all full length myosin members and expressed in protoplasts, revealed specific localization and movement among members. In this time, myosins were expressed in physiologically more intact environment, cultured cells and transgenic plants of Arabidopsis. In addition, truncated tail domain was expressed, acting as dominant negative because it masks myosin binding site. As a result, tail domain of some members show different localization from that of the full length. Indicating, myosin members have independent functions in particular trafficking pathway. From these results, functions and roles of myosin members in intracellular trafficking will discuss.

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In Arabidopsis, myosin VIII class consists of four members: ATM1, ATM2, VIIIA and VIIIB. There were several reports that tail domains of myosin VIII were localized at plasmodesmata, cell nuclei, endoplasmic reticulum and endosomes, suggesting that tail domains were diversified among myosin VIII members and each of them could associate with specific proteins. To elucidate the mechanism for each myosin VIII, we investigated the subcellular localization of full length myosin VIII which were transiently expressed in protoplasts and cultured cells. Myosin ATM1 and VIIIA were localized at the plasmodesmata, and in dot-like structures. Myosin ATM2 located on the dispersed dots and in cell nuclei. Myosin VIIIB was localized in condensed membranous structures. Next, we searched proteins that specifically interact with myosin VIII tail domains by pull down analysis. We will discuss the function of candidate molecules as the adaptor proteins of myosin VIII members.

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In animal cells, RAB5 GTPase is known as a molecular switch regulating various aspects of endosomal functions including membrane fusion, motility, and alteration of lipid composition. RAB5 fulfills these diverse functions through interactions with various effector molecules. In Arabidopsis thaliana, there are two conventional type of RAB5 members, ARA7 and RHA1. ara7 rha1 double mutants show gametophytic lethality, which indicates their redundant but essential function. The signal from plant RAB5s could also be mediated via effector molecules to induce downstream reactions, but no such effectors have been identified thus far. We looked for effectors of A. thaliana RAB5s by a yeast two-hybrid method. As a result, we succeeded in isolation of one candidate whose subcellular localization was changed depending on the nucleotide state of ARA7. The close relationship between the candidate and plant RAB5s was also supported by genetic studies. Surprisingly, the effector candidate also interacted with the other endosomal RAB. In this meeting, we will report results of biochemical and genetic studies on the effector candidate.

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Rab GTPase is one of the key regulators in membrane trafficking. Rab11, a broadly conserved Rab GTPase group in eukaryotes, has evolved in a unique way in plants. Plant Rab11 has notable diversity, while yeast and animals possess a few members. In Arabidopsis thaliana, 57 Rab GTPases are encoded in its genome in total, 26 of which are classified into the Rab11 group (subclassified into RabA1-RabA6). It is already known that animal Rab11 plays an important role in regulating various cellular functions. On the other hand, the precise function of plant Rab11 remains largely unknown. To reveal the plant Rab11 function, we analyzed subcellular localization of 9 members belonging to the RabA1 subgroup (RabA1a-RabA1i). We have already reported that RabA1e located on the mobile punctate structures and accumulated in the tip region of root hair cells. In this study, we performed the precise analysis of the subcellular localization of RabA1e, and found that RabA1e is almost co-localized with R-SNARE protein which functions in secretory pathway. We will also report about the novel interacting proteins with RabA1 members.

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The RAB GTPase, including RAB5, is known to act as a molecular switch through cycling between GTP-bound and GDP-bound states. The GTP-bound state is considered as the active form to which effector molecules specifically interact. In mammalian cells, RAB5 effectors are known to trigger various downstream phenomena. RAB5 is conserved in a broad range of eukaryotic organisms including plants. In addition to the conventional-type RAB5 homologs, land plants possess plant-specific RAB5 homologs (ARA6 in Arabidopsis thaliana) (Ueda et al., 2001). Our previous studies indicated that ARA6 and conventional-type RAB5 regulate plant-specific functions by coordinating different membrane traffic pathways. This regulatory system is thought to involve RAB5 effector molecules; however, the plant RAB5 effectors are hitherto unknown. To elucidate the molecular basis of such system, we focused on ARA6 and identified its effectors. In this meeting, the cell biological and physiological significance of PUF2 (Plant-unique RAB5 Effector 2)-ARA6 interaction will be discussed.

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Post-Golgi organelles play fundamental roles in various plant functions of higher order. However, knowledge on the molecular mechanisms of their biogenesis and trafficking system interconnecting them is still limited. We are studying molecular mechanisms of endocytosis and vacuolar transport and how these trafficking pathways participate in plant morphogenesis with a special focus on plant-unique RAB and SNARE molecules. A. thaliana has two types of RAB5 members, conventional RAB5 and plant unique ARA6. Distinct subcellular localization of ARA6 from conventional RAB5 suggested functional differentiation between these two RAB5 groups, but their precise functions remained unknown. On the other hand, VAMP727 is a plant-unique R-SNARE, which is characterized by an insertion of 20 amino acids in its N-terminal longin domain. We have already reported that VAMP727 forms a SANRE complex with VAM3, VTI11, and SYP51 on a subpopulation of PVCs closely associated with the vacuolar membrane (Ebine et al., 2008). In this meeting, we will report our recent results on a regulatory role of ARA6 in VAMP727 complex formation.

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SNARE molecules are key regulators of vesicular traffic, executing membrane fusion between transport vesicles or organelles and target membranes. These molecules are classified into four main types (Qa, Qb, Qc and R) based on the structural property of their coiled-coil region, called SNARE motif. One of major plant R-SNARE families, VAMP7, consists of two subgroups, VAMP71 and VAMP72. In seed plants, structurally distinct VAMP72 molecules harboring an acidic cluster comprising about 20 amino acids in the longin domain are also conserved. We previously reported that this type of VAMP72 molecule in Arabidopsis thaliana, VAMP727, is involved in membrane trafficking around endosomes. To clarify the functional relevance of the acidic cluster, we investigated the effects of deletion and artificial insertion of the acidic cluster. Our results indicated that the acidic cluster played a critical role for functional conversion of VAMP72 members during plant evolution.

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Arabidopsis VAM3/SYP22, one of the SYP2 family SNARE proteins, 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). Previously, we reported that vam3 mutants exhibited the phenotypes of wavy leaves, delayed growth, semi-dwarfism and abnormal distribution of myrosin cells (1). Although PEP12/SYP21 and PLP/SYP23 are homologues to VAM3/SYP22, the physiological function of PEP12/SYP21 and PLP/SYP23 in plants remains unknown. To elucidate a role of PEP12/SYP21 and PLP/SYP23 in protein trafficking and development of plant cells, we generated double and triple mutants of the SYP2 family SNARE proteins of Arabidopsis thaliana. By analyzing the phenotypes of these mutants, we found the functional redundancy between these three SYP2 family SNARE proteins. We will discuss about the functions of SYP2 family SNARE proteins in higher plants.
(1) Shirakawa, M. et al, Plant Cell Physiol., 50, 1319-1328 (2009).

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MAG3 is involved in intracellular transport of seed storage protein.
Seed storage proteins are synthesized on the endoplasmic reticulum (ER) as precursors and then transported to protein storage vacuoles, where they are processed into mature forms. Here, we isolated and characterized an Arabidopsis mutant, maigo3 (mag3), that accumulated the precursors of storage proteins in dry seeds. mag3 seed cells contained a number of novel structures with electron-dense cores. Because these structures were surrounded by ribosomes, they seem to be derived from ER. MAG3 gene encodes an unknown protein, which contains a number of coiled-coil regions and one transmembrane domain at the C-terminus. Biochemical analysis revealed that MAG3 protein localized on the ER membrane, and that the transmembrane domain is responsible for ER localization and function of MAG3 protein. Our findings suggest that MAG3 protein is involved in the protein transport between ER-Golgi complex. Functional model of MAG3 will be discussed. 944
Reference: Li et al. (2006) Plant Cell, 18, 3535-3547.

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The endoplasmic reticulum (ER) forms a dynamic polygonal network composed of tubules, sheets, and three-way junctions. Although the complex ER morphology supports the diverse cellular functions, the molecular mechanisms responsible for the organization of these structures are obscure. Here we report the isolation and characterization of mutants of Arabidopsis thaliana that are defective in ER morphology (ermo1, ermo2, and ermo3). Among three mutants, ermo1 and ermo2, which showed partially similar phenotypes, were defective in GNOM-LIKE1 (GNL1) and SEC24a, respectively (1). Both GNL1/ERMO1 and SEC24a/ERMO2 were thought to be involved in the ER-Golgi transport. Our results suggests that the unknown cargo proteins that are specifically transported by GNL1/ERMO1 and SEC24a/ERMO2 may have crucial roles. On the other hand, we identified the responsible gene for ermo3 to be a member of GDSL-motif lipase family, which is predicted to be localized within the vacuoles. Our findings suggest a novel pathway that connects ER morphology to lipid metabolic processes.
(1) Nakano, R.T. et al., Plant Cell,10.1105/tpc.109.068270 (2009).

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The endoplasmic reticulum (ER) has an elaborate quality control system, that ensures that only correctly folded proteins are transported to the Golgi apparatus. In mammalian cells, ERdj3, an ER resident Hsp40 co-chaperone, functions in the ER quality control (ERQC) as a partner for BiP. We identified an Arabidopsis ortholog of ERdj3, AtERdj3B, and showed AtERdj3B also functions in the ERQC. T-DNA mutants of the AtERDJ3B gene were viable and did not show any growth or developmental defects. However, the aterdj3b mutants became sterile when they grew at high temperature. This was mainly due to that the release of pollen grains from the mutant anthers. Analyses of microscopy observation revealed abortion and collapse of pollen in the mutant anthers. Those defects were not suppressed by expression of the aterdj3b(H54Q) mutant protein, which is defective in the interaction with BiP, indicating that the AtERdj3B-BiP interaction is essential. An ER protein, UGGT, functions in the quality control of glycoproteins in the ER, and its mutant also became sterile when it was grown at high temperature. These results suggest that the ERQC is essential for pollen fertility at high temperature.

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Binding protein (BiP) is one of the most important key chaperons for protein folding in endoplasmic reticulum (ER) lumen. Increasing of mis-folded or incorrectly translated proteins in ER promptly induces BiP gene expression as unfold protein response (UPR). To investigate the role of BiP during rice seed maturation, we generated transgenic rice plants in which the rice BiP gene (AK065743) was overexpressed or suppressed in a seed specific manner. Both seeds of BiP-overexpression and suppression lines displayed a chalky phenotype as flowly and shurunken feature. Furthermore, protein sorting and accumulation systems significantly perturbed in their endosperm cells. RT-PCR analyses indicated that many chaperone genes, other BiP, HSP70, calnexin, Calreticulin and PDI genes, were increased their expression in both of transgenic seeds. These results suggested that transgenic rice seeds with either much higher or lower expression of BiP have caused ER stress in their endosperm cells. Furthermore, immuno-blot analyses of the transgenic rice seed suggested that changes of BiP level significantly affects a quality control system in endosperm cells.

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The messenger RNA of the rice seed storage protein prolamine is targeted to the endoplasmic reticulum (ER) membranes surrounding prolamine protein bodies via a mechanism which is dependent on both RNA sorting signals and the actin cytoskeleton. In this study we have used an RNA bait corresponding to the previously characterized 5'CDS prolamine zipcode sequence for the capture of RNA binding proteins (RBPs) from cytoskeleton-enriched fractions of developing rice seed. In comparison to a control RNA, the zipcode RNA bait sequence captured a much larger number of proteins, eighteen of which have been identified by tandem mass spectrometry. Western blots demonstrate that several of the candidate proteins analysed to date show good to excellent specificity for binding to zipcode over non-zipcode RNA bait. The protein expression profile of RBP-A, an hnRNP, closely follows prolamine gene expression and in addition to being present in nuclei, RBP-A also co-localizes with microtubules and is associated with cortical ER membranes. Immunoprecipitation studies show that RBP-A is bound to prolamine and glutelin RNAs in vivo, supporting a direct role in storage protein gene expression.

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To identify the function of genes that regulate the processing of proglutelin, we performed an analysis of glup3 mutants that accumulate excess amounts of proglutelin and lack the vacuolar processing enzyme (VPE). VPE activity in developing seeds from glup3 lines was reduced remarkably compared to the wild type. Microscopic observations showed that α-globulin and proglutelin were distributed homogeneously within glup3 protein storage vacuoles (PSVs), and that glup3 PSVs lacked the crystalline lattice structure typical of wild type PSVs. This suggests that the processing of proglutelin by VPE in rice is essential for proper PSV structure and compartmentalization of storage proteins. Growth retardation in glup3 seedlings was also observed, indicating that the processing of proglutelin influences early seedling development. These findings indicate that storage of glutelin in its mature form as a crystalline structure in PSVs is required for the rapid use of glutelin as a source of amino acids during early seedling development. In conclusion, VPE plays an important role in the formation of protein crystalline structures in PSV.

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We investigate the function of nucleotide pyrophosphatase/phosphodiesterase (NPP) in rice. Six NPP isozyme genes have been found in rice. Previously, we purified and characterized NPP1, 2 and 6. NPP1 and 6 were divided into ADP-glucose hydrolyzing type, while NPP2 was nucleotide hydrolyzing type. All NPP isozymes were conjugated with Con A-recognized N-glycans. Furthermore, the expression and targeting of NPP-GFP fusion genes revealed that more than 70% of NPPs localize within the plastid. In the present study, we used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) techniques exoglycosidase digestions of plastid localized glycoprotein NPP1,to identify N-glycan structures. N-glycan structures were identified 27 types and contained high mannose-type (14.9%), complex-type (53.8 %), and Pauci-mannose-type N-glycans (31.3%).
The overall results suggested that glycoprotein NPP1 targeting to plastid and biogenesis was involved in the trafficking of glycosylated proteins from the endoplasmic reticulum–Golgi system.

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Sec-translocase is an evolutionarily conserved protein translocation pathway and its functions are protein translocations, secretions and insertions. SecA,a motor protein, plays the important roles in translocations and couples ATP hydrolyzed energy with protein translocations. There is SecA homologue in plants, it functions in translocation from stroma into thylakoid lumen. Mycobacteria and Listeria possess two SecAs that distinguish its substrate proteins. But these substrates-distinction mechanisms are not known in plant cells.
Analysis of the complete genome sequences of a unicellular red alga Cyanidioschyzon merolae revealed two secA genes were encoded in the nuclear and plastid genomes respectively. These two secA genes were transcribed, and we suggested they possess some functions. These secAs were distinct phylogenetically. We demonstrated the homogeneous protein interactions in the nuclear and the plastid-encoded SecAs, and the heterogeneous interaction between the nuclear-encoded and the plastid-encoded SecA. And we demonstrated the optimal temperatures of its ATPase activities were obviously distinct. We discuss temperatures-distinction mechanism of C. merolae SecAs.

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Rubisco, CO₂ fixing enzyme, is the most abundant leaf protein accounts for 15-30% leaf nitrogen in C3 plants. Despite its abundance, Rubisco is rate-limiting factor under conditions of saturated light and ambient CO₂ concentration. Rubisco is a decahexamer comprised of eight small subunit coded by RBCS in nuclear genome and eight large subunits encoded by RBCL in plastome. In higher plants, RBCS forms a multigene family but its biological function is still unknown. In this study, RNAi-mediated knock-down transformants of individual RBCS were generated in rice (Oryza sativa L.) and their leaf Rubisco contents were determined to evaluate their contribution to leaf Rubisco contents. Out of five RBCS in rice, namely, OsRBCS1 to 5, OsRBCS2 to 5 were knocked-down since they were highly expressed in leaf blades. In T₀ generation of transformants, the mRNA levels of RNAi-targeted RBCS severely declined without a large effect on those of other RBCS genes. Rubisco contents of OsRBCS2, 3, 4 and 5 knock-down transformant declined by 35, 25, 16 and 32% in comparison with wild-type, respectively. These results suggest that OsRBCS2 to 5 are all responsible for Rubisco synthesis in rice.

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Rubisco is a rate-limiting factor for light-saturated photosynthesis under the present atmospheric CO₂ pressures. Additionally, Rubisco is the most abundant leaf protein in C₃ species. Therefore, Rubisco plays an important role in both photosynthesis and N economy in plant growth. However, Rubisco does not limit photosynthesis under elevated CO₂ levels and the amount of Rubisco protein is clearly excessive under conditions of CO₂ enrichment. We produced a series of transgenic rice plants with decreased Rubisco content by RNAi technology. OsRBCS3-1-1 and OsRBCS3-3-6, which have 70% wild type Rubisco, are obtained as the transformants with optimal Rubisco content for CO₂-saturated photosynthesis. We investigated growth of these transformants under ambient conditions. Plant height, leaf age and tiller number were not significantly different between WT and transformants. The biomass of transformants was smaller than that of WT at 70d. This decrease in biomass in transformants was due to a significant decrease in RGR between d42 and 70.

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Plant fructose-1,6-bisphosphate aldolase (FBA) is an essential enzyme for glycolysis and Calvin cycle, and FBAs are classfied into cytosolic- and plastidic-type. Three plastidic FBA isozymes were discovered on the Arabidopsis genome, which are designed as FBA1 (At2g01140), FBA2 (At2g21330) and FBA3 (At4g38970). Based on amino acid sequences from various plants, those isozymes can be classified into FBA1 and FBA2/3 group. We previously reported that the Arabidopsis FBA1 was identified as a glutathionylated protein and that it played a central role in a glutathione-dependent regulation of photosynthesis. In this study, we newly made transgenic Arabidopsis plants overexpressing each plastidic FBA isozyme in order to elucidate their enzymatic properties. We obtained transformants that accumulated plastidic FBA proteins more than three times of wild type level, so that FBA activity was relevantly enhanced. Native-PAGE analysis using an extract from the leaf showed that overexpression of each FBA isozyme had an effect on the characteristic of FBA tetramer. Based on the result, we will discuss the characteristic and significance of the plastidic FBA isozymes.

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We have reported that plastidic fructose-1,6-bisphosphate aldolase (FBA) is in vivo glutathionylated and that this isozyme, FBA1, is regulated by glutathione and important for CO₂ fixation. Arabidopsis plants have another two FBA isozymes (FBA2 and FBA3) in chloroplasts. Thus, to elucidate the function of FBA1 in chloroplasts, we performed functional analysis of Arabidopsis mutants of each FBA isozyme. FBA1, FBA2 and FBA3 constituted 2%, 28% and 70%, respectively, of the total plastidic FBA protein in wild-type leaves. Compared to wild-type plants, the fba1-1 mutant with 40% decreased FBA1 exhibited reduced sugar accumulation and CO₂ assimilation. These phenotypes were more severe in the fba1-4 mutant with truncation of the carboxyl terminal amino acid residues in the FBA1 protein. T-DNA insertion of FBA3 restricted sugar accumulation but did not CO₂ assimilation. There was no difference in these phenotypes between wild-type plant and T-DNA insertional mutant of FBA2. These results indicate that the FBA1 plays an important role in maintenance of chloroplasts. Based on these findings, we will discuss the function of FBA1 in the Calvin cycle.

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CO₂ assimilation by cyanobacteria is very efficient because of the operation of the CO₂-concentrating mechanism (CCM), which is activated in response to CO₂ deficiency. In Synechococcus elongatus PCC 7942, a LysR-type transcriptional regulator CmpR regulates transcription of the CCM-related genes, including the cmpABCD operon, the sbtA gene, and the ndhF3D3chpY operon. CmpR is similar to bacterial CbbR proteins, which is known to recognize and bind to the CbbR motif (TNA-N_7/8-TNA). The regulatory region of cmpABCD is 950 bp long and has been shown by gel shift assay to have several binding sites for CmpR. Moreover, this region contains seven CbbR-binding motifs (cmp1-cmp7), mutations in three of which (cmp3, 5, 6) inhibit the low-CO₂ response of the promoter, suggesting CmpR binding to these sites. In this study, we investigated the binding of CmpR to the DNA fragments carrying cmp3, 5 and 6, respectively. Surprisingly, modification of cmp3 and 6 did not affect the CmpR binding, indicating that CmpR can bind to DNA sequences other than those containing the CbbR motif.

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Marine diatoms can concentrate inorganic carbon (Ci) under air level pCO₂. Accumulated Ci is thought to be delivered to Rubisco efficiently catalysed by internal carbonic anhydrases (CAs). In the marine diatom Phaeodactylum tricornutum, two chloroplastic CAs, PtCA1 and 2, were so far identified and its localization in the pyrernoid is strongly suggested. Other 6 putative cas are found in P. tricornutum genome but their localization is unknown. It's also suggested C₄ metabolism is a part of the diatom CCMs. If this is the case in P. tricornutum, PEPC, a HCO³_- fixation enzyme, most probably locate in the cytosol and, PEPCK or ME, decarboxylation enzymes, need to locate in the chloroplast. In the present study, putative CAs and C4 genes were cloned from P. tricornutum cDNA library. Expressions of these genes were analyzed by RT-PCR. These 6 putative cas and C₄ component genes were fused with egfp and induced into the cells of P. tricornutum. As a result, all 6 putative CAs were localized at the four-layered membrane systems surrounding the chloroplast. The mechanisms how diatom cells control the Ci flux inside the cytosol and the chloroplast will be discussed from these results.

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Coccolithophorids (Haptophyta) acquired their chloroplasts via the secondary endosymbiosis and thus possess four-layer envelopes. In a coccolithophorid, Emiliania huxleyi, we already reported that C₄ compounds is photosynthetically produced via anaplerotic β-carboxylation reactions in addition to the C₃ cycle. Pyruvate carboxylase (PYC) was suggested to play an important role in the production of C₄ compound since PYC transcript remarkably increased under illumination. Based on the prediction of N-terminal targeting signal, PYC is expected to function in the chloroplast. In the present study, we could successfully detect PYC activity in the crude extract of E. huxleyi. The PYC activity was completely inhibited by the addition of avidin, a specific inhibitor of biotin carboxylases including PYC. Therefore, the activity we detected is actually derived from PYC, not from other β-carboxylation enzymes such as phosphoenolpyruvate carboxylase. Using immunofluorescence labeling, we showed that E. huxleyi PYC locates in the chloroplast. These data suggest the contribution of chloroplastic PYC to the active β-carboxylation during photosynthesis in E. huxleyi.

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Most of higher plants have leaves with adaxial-abaxial polarity. Adaxial region is specified by adaxial-specific genes, whereas abaxial region is specified by abaxial-specific genes. However, it is still unknown how the expression patterns of adaxial-specific or abaxial-specific genes are established.
We analyzed novel adaxial-abaxial mutant, enlarged fil expression domain1 (enf1). enf1 mutant had some leaves which lost robustness of adaxial-abasial polarity. ENF1 gene encoded SUCCINIC SEMIALDEHYDE DEHYDEROGENASE (SSADH), which is one of GABA shunt enzymes. We analyzed another mutant of GABA shunt enzymes, gabat1> mutant. gabat1> mutant showed weak abaxializing phenotype and suppressed enf1. We will discuss how GABA shunt regulates the polarity along adaxial-abaxial axis.

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