中井 朋則

Chloroplast-actin (cp-actin) filaments are crucial for light-induced chloroplast movement, and appear in the front region of moving chloroplasts when visualized using GFP-mouse Talin. They are short and thick, exist between a chloroplast and the plasma membrane, and move actively and rapidly compared to cytoplasmic long actin filaments that run through a cell. The average period during which a cp-actin filament was observed at the same position was less than 0.5 s. The average lengths of the cp-actin filaments calculated from those at the front region of the moving chloroplast and those around the chloroplast periphery after stopping the movement were almost the same, approximately 0.8 µm. Each cp-actin filament is shown as a dotted line consisting of 4-5 dots. The vector sum of cp-actin filaments in a moving chloroplast is parallel to the moving direction of the chloroplast, suggesting that the direction of chloroplast movement is regulated by the vector sum of cp-actin filaments. However, once the chloroplasts stopped moving, the vector sum of the cp-actin filaments around the chloroplast periphery was close to zero, indicating that the direction of movement was undecided. To determine the precise structure of cp-actin filaments under electron microscopy, Arabidopsis leaves and fern Adiantum capillus-veneris gametophytes were frozen using a high-pressure freezer, and observed under electron microscopy. However, no bundled microfilaments were found, suggesting that the cp-actin filaments were unstable even under high-pressure freezing.

In vitro reconstitution of whole cellular events is one of the important goals in synthetic biology. Using a cell-free protein synthesis (CFPS) system reconstituted with human translation factors and chaperones, we reproduced the biogenesis of β-actin, synthesis, folding, and polymerization in a test tube. This system enabled us to define which step of the β-actin biogenesis was defective in genetic mutations related to diseases. Hence, the CFPS system reconstituted with human factors may be a useful tool for analyzing proteostasis in eukaryotes.

X-ray micro-CT is one of the most useful techniques to examine 3D cellular architecture inside dry seeds. However, the examination of imbibed seeds is difficult because immersion in water causes a decline in the image quality. Here, we examined the use of ionic liquids for specimen preparation of chemically fixed imbibed seeds of Arabidopsis. We found that treatment with high concentrations of ionic liquids after osmium tetroxide fixation helped not only to prevent the structural damage caused by seed shrinkage, but also to preserve the image quality. Under these conditions, the cellular architecture of seeds was also well maintained.

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global–local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global–local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.

<p>Insertion of a sheet of cover glass in the lid of a glass-based dish is a useful technique for observation by differential interference contrast microscopy. However, microscopic observations of algal cultures using this glass-based dish are often interfered with by dew condensation on the glass surface of the lid when the culture is incubated for a long time. To prevent dew development on the lid surface during incubation, we employed a glass-based dish whose inner surface of the glass-lid is coated with 2-methacryloyloxyethyl phosphorylcholine polymer. Using this modified dish, we could observe thalli development of Coleochaete scutata successively for four weeks without dew condensation on the lid.</p>

In order to find out how plants maintain cell surfaces in a state that allows the rapid increase in surface area and volume of cells during imbibition, we examined the embryo cell shape in dry seeds of Glycine max cv. Hakuchou, Lotus miyakojimae and Arabidopsis thaliana. SEM observations showed that cell walls in dry seeds were often folded (see outline of the yellow-colored cell in Fig. 1A) although they were flat in imbibed seeds (Fig. 1B). To investigate whether the folded cell walls in the dry seeds really exist or not, we further examined seeds in vivo using X-ray micro-CT equipment in BL47XU at SPring-8 and also observed chemically fixed, plastic embedded seeds by high-voltage electron tomography. Our observations suggest that folded cell walls in dry seed embryos are a common structure.

Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 mu m. Treatments with cytoskeletal inhibitors for 15min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.

© 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry.The expression of the gene for a proteinase (Rep1) is upregulated by gibberellins. The CAACTC regulatory element (CARE) of the Rep1 promoter is involved in the gibberellin response. We isolated a cDNA for a CARE-binding protein containing a Myb domain in its carboxyl-terminal region and designated the gene Carboxyl-terminal Myb1 (CTMyb1). This gene encodes two polypeptides of two distinctive lengths, CTMyb1L and CTMyb1S, which include or exclude 213 N-terminal amino acid residues, respectively. CTMyb1S transactivated the Rep1 promoter in the presence of OsGAMyb, but not CTMyb1L. We observed an interaction between CTMyb1S and the rice prolamin box-binding factor (RPBF). A bimolecular fluorescence complex analysis detected the CTMyb1S and RPBF complex in the nucleus, but not the CTMyb1L and RPBF complex. The results suggest that the arrangement of the transfactors is involved in gibberellin-inducible expression of Rep1.

Cellulases are enzymes that normally digest cellulose; however, some are known to play essential roles in cellulose biosynthesis. Although some endogenous cellulases of plants and cellulose-producing bacteria are reportedly involved in cellulose production, their functions in cellulose production are unknown. In this study, we demonstrated that disruption of the cellulase (carboxymethylcellulase) gene causes irregular packing of de novo-synthesized fibrils in Gluconacetobacter xylinus, a cellulose-producing bacterium. Cellulose production was remarkably reduced and small amounts of particulate material were accumulated in the culture of a cmcax-disrupted G. xylinus strain (F2-2). The particulate material was shown to contain cellulose by both solid-state C-13 nuclear magnetic resonance analysis and Fourier transform infrared spectroscopy analysis. Electron microscopy revealed that the cellulose fibrils produced by the F2-2 cells were highly twisted compared with those produced by control cells. This hypertwisting of the fibrils may reduce cellulose synthesis in the F2-2 strains.

Gibberellins (GAs) are involved in the expression of cysteine proteinase genes in germinated cotyledons of common bean seeds. Because DELLA proteins are known to be transcriptional repressors mediating GA signaling, we isolated two cDNA clones encoding DELLA proteins (PvGAI1 and PvGAI2) from common bean seedlings to examine the mechanism of GA signaling involved in the expression of the proteinase genes. RT-PCR and RNA blot analyses indicated that the level of mRNA in germinated cotyledons was higher for PvGAI2 than for PvGAI1. We also found that transient expression of PvGAI2, but not that of PvGAI1, repressed the promoter activities of GA-inducible cysteine proteinase genes, EP-C1 and CP2, in germinated cotyledons. These findings suggest that PvGAI2 is mainly responsible for regulating the expression of proteinase genes in germinated cotyledons. Application of a GA-biosynthesis inhibitor, prohexadione calcium to common bean seeds had little effect on the RNA level of PvGAI2, although the inhibitor repressed genes for EP-C1 and CP2. Because it is known that GA induces degradation of DELLA proteins, our findings suggest that the level of GA, but not the mRNA expression of PvGAI2, regulates the protein level of PvGAI2 suppressing the proteinases genes in germinated cotyledons.

Bacillus brevis (Brevibacillus parabrevis) ATCC 8185 synthesizes two kinds of antibiotic peptides, cyclopeptide tyrocidine and linear gramicidin. The production of linear gramicidin can be induced by the standard method (using a skim milk medium for pre-culture and beef broth for the main culture) employed for the induction of tyrocidine. In this study, we tried to determine the optimal growth medium for B. brevis ATCC 8185 for synthesizing linear gramicidin. The yield of linear gramicidin produced by the standard method was 3.11 μg/ml. When beef broth was used both as the pre-medium and the main medium, the yield of the antibiotic was only 0.59 μg/ml. To confirm the influence of skim milk, the strain was grown in a 1% skim milk medium. As a result, the amount of linear gramicidin produced reached 20.3 μg/ml. These findings show the importance of skim milk in the production of linear gramicidin. In the skim milk medium, the cells produced an extracellular protease 2h before the linear gramicidin was expressed. The 1% skim milk medium pretreated by this protease did not allow the induction of linear gramicidin into the cells, and protease activity was not detected in the supernatant of the culture. When the cells were cultivated in a 1% egg albumin medium, protease activity from the supernatant of the culture was detected, but production of linear gramicidin was not observed. Therefore, a 1% casein medium was used for production of linear gramicidin. As a result, the yield of linear gramicidin produced in the medium reached 6.69 μg/ml. We concluded that a digested product of the extracellular protease from casein enhances linear gramicidin production.

An ORF2gene located upstream of the cellulose synthase(hcs)operon of Acetobacter xylinum BPR2001 was disrupted and a mutant (M2-2) was constructed. In static cultivation, the parent strain produced a tough, colorless, and insoluble cellulose pellicle, whereas M2-2 culture produced a thin, yellow, and fragile pellicle. The results of X-ray diffraction and C-13 solid-state NMR indicated that the product of M2-2 is a mixture of cellulose I, cellulose II, and amorphous cellulose. The cellulose I to cellulose II ratio of the mixture was evaluated from the signal areas of C6 to be about 1:2. Electron microscopy revealed that the product of M2-2 included ribbon-like cellulose and irregularly shaped particles attached to the ribbons. Oil the other hand, the mutant complemented with plasmid pSA-ORF2/k containing the ORF2 gene and BPR2001 produced only cellulose 1. These results indicate that the ORF2 gene is involved in the production and crystallization of cellulose 1 microfibrils by this microorganism. (C) 2002 Elsevier Science (USA). All rights reserved.

Acetan is a water-soluble polysaccharide produced by a bacterial cellulose (BC) producer, Acetobacter xylinum. An acetan-nonproducing mutant, EP1, was generated from wild-type A. xylinum BPR2001 by the disruption of aceA, which may act to catalyze the first step of the acetan biosynthetic pathway in this bacterium. EP1 produced less BC than the wild-type strain. However, when EP1 was cultured in a medium containing acetan, BC production was stimulated and the final yield of BC was equivalent to that of BPR2001. The culture broth containing acetan was more viscous and the free cell number was higher than that of the broth without the polysaccharide, so acetan may hinder the coagulation of BC in the broth. The addition of 1.5 g/l agar also increased BC production we concluded that acetan and BC syntheses were not directly related on the genetic level. © 2002 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.

The 5' upstream region (about 3.1 kb) of the cellulose synthase operon (bcs operon) has been isolated by cloning from Acetobacter xylinum strain BPR 2001. The expression level of the upstream region was determined using sucrose synthase cDNA as a reporter gene in the shuttle vector pSA19. The expression occurred with the 1.1-kb upstream sequence from the ATG start codon of the bcs operon but not with the 241-bp upstream sequence in A. xylinum, although neither the 1.1-kb nor the 241-bp upstream sequence caused any expression as a promoter in Escherichia coli. The level of expression with the I.l-kb upstream sequence in A. aceti was 75% of that in A. xylinum. These results suggest that the upstream region functions as a specific promoter for the Acetobacter genus. The expression was reduced by the introduction of the 241-bp upstream region between the lac promoter and the reporter gene in E. coli and was not detected in A. xylinum. This suggests that the short upstream region composed of 241 bp contains the site(s) which causes a negative regulation on the transcription for bcs operon. The production of recombinant protein with the ribosome-binding site (RBS) of A. xylinum obtained from the bcs operon, was reduced to about half in E. coli, and that with the site of the lac promoter was also reduced to about half in A. xylinum. This shows that a species-specific predominance occurs during interaction between mRNA and 16S rRNA in the RES between A..xylinum and E. coli. (C) 1998 Elsevier Science B.V. All rights reserved.

About 80% of radioactivity was recovered in asymmetrically labeled sucrose from UDP-[C-14]glucose or [C-14]fructose with recombinant mung bean sucrose synthase expressed in Escherichia coli harboring pEB-01. This high recovery is due to the fact that the enzyme conserving the activity of sucrose synthase has a similar affinity for UDP-glucose and fructose to an intact enzyme from the mung bean, but a lower affinity for sucrose.

An open reading frame was found 214 bp downstream of the cellulose synthase operon of Acetobacter. The encoded amino acid sequence was found to be similar to some beta-glucosidases (G3ases), We detected G3ase activity in the culture medium and analysis of the N-terminal amino acid sequence showed that this gene encodes the enzyme, Therefore, it is possible that this region is a gene cluster for cellulose synthesis.

The cDNA fragment coding for mung bean (Vigna radiata Wilczek) sucrose synthase was introduced into the expression vector pET-20b resulting in the construction of plasmid pEB-01. After transformation of Escherichia coli strain BL21(DE3) cells by pEB-01 and induction with isopropyl thio-beta-galactoside, high level expression of the recombinant enzyme was obtained. The enzyme had a tetrameric form that conserved the activity of sucrose synthase. Although the K-m and V-max of the recombinant enzyme acting on either UDP-glucose or fructose were very close to those of the native enzyme isolated from mung bean seedlings, the K-m for sucrose was higher by a factor of 10 for the recombinant enzyme. This suggests that the recombinant sucrose synthase has a tendency to synthesize sucrose, although the native enzyme catalyzes a freely reversible reaction.