Cell-free translation system with artificial lipid-monolayer particles as a unique tool for characterizing lipid-monolayer binding proteins.

Methods for functional analysis of proteins specifically localizing to lipid monolayers such as rubber particles and lipid droplets are limited. We have succeeded in establishing a system in which artificially prepared lipid monolayer particles are added to a cell-free translation system to confirm the properties of proteins that specifically bind to lipid monolayers in a translation-coupled manner.

The NAD Kinase Slr0400 Functions as a Growth Repressor in Synechocystis sp. PCC 6803.

NADP+, the phosphorylated form of nicotinamide adenine dinucleotide (NAD), plays an essential role in many cellular processes. NAD kinase (NADK), which is conserved in all living organisms, catalyzes the phosphorylation of NAD+ to NADP+. However, the physiological role of phosphorylation of NAD+ to NADP+ in the cyanobacterium Synechocystis remains unclear. In this study, we report that slr0400, an NADK-encoding gene in Synechocystis, functions as a growth repressor under light-activated heterotrophic growth conditions and light and dark cycle conditions in the presence of glucose. We show, via characterization of NAD(P)(H) content and enzyme activity, that NAD+ accumulation in slr0400-deficient mutant results in the unsuppressed activity of glycolysis and tricarboxylic acid (TCA) cycle enzymes. In determining whether Slr0400 functions as a typical NADK, we found that constitutive expression of slr0400 in an Arabidopsis nadk2-mutant background complements the pale-green phenotype. Moreover, to determine the physiological background behind the growth advantage of mutants lacking slr04000, we investigated the photobleaching phenotype of slr0400-deficient mutant under high-light conditions. Photosynthetic analysis found in the slr0400-deficient mutant resulted from malfunctions in the Photosystem II (PSII) photosynthetic machinery. Overall, our results suggest that NADP(H)/NAD(H) maintenance by slr0400 plays a significant role in modulating glycolysis and the TCA cycle to repress the growth rate and maintain the photosynthetic capacity.

One of the NAD kinases, sll1415, is required for the glucose metabolism of Synechocystis sp. PCC 6803.

Pyridine nucleotides (NAD(P)(H)) are electron carriers that are the driving forces in various metabolic pathways. Phosphorylation of NAD(H) to NADP(H) is performed by the enzyme NAD kinase (NADK). Synechocystis sp. PCC 6803 harbors two genes (sll1415 and slr0400) that encode proteins with NADK homology. When genetic mutants for sll1415 and slr0400 (Δ1415 and Δ0400, respectively) were cultured under photoheterotrophic growth conditions only the Δ1415 cells showed a growth defect. In wild-type cells, the sll1415 transcript accumulated after the cells were transferred to photoheterotrophic conditions. Furthermore, NAD(P)(H) measurements demonstrated that a dynamic metabolic conversion was implemented during the adaptation from photoautotrophic to photoheterotrophic conditions. Electron microscopy observation and biochemistry quantification demonstrated the accumulation of glycogen in the Δ1415 cells under photoheterotrophic conditions at 96 h. Quantitative real-time reverse transcription PCR (qRT-PCR) demonstrated the accumulation of mRNAs that encoded glycogen biosynthesis-related enzymes in photoheterotrophic Δ1415 cells. At 96 h, enzyme activity measurement in the photoheterotrophic Δ1415 cells demonstrated that the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were decreased, but the activities of glucose dehydrogenase were increased. Furthermore, metabolomics analysis demonstrated that the Δ1415 cells showed increased glucose-6-phosphate and 6-phosphogluconate content at 96 h. Therefore, sll1415 has a significant function in the oxidative pentose phosphate (OPP) pathway for catabolism of glucose under photoheterotrophic conditions. Additionally, it is presumed that the slr0400 had a different role in glucose catabolism during growth. These results suggest that the two Synechocystis sp. PCC 6803 NADKs (Sll1415 and Slr0400) have distinct functions in photoheterotrophic cyanobacterial metabolism.

Effects of inactivation of the cyAbrB2 transcription factor together with glycogen synthesis on cellular metabolism and free fatty acid production in the cyanobacterium Synechocystis sp. PCC 6803.

Deletion of the cyAbrB2 (Sll0822) transcription factor in Synechocystis sp. PCC 6803 causes aberrant accumulation of glycogen. We previously tried to redirect the excess carbon stored as glycogen in the cyabrB2-disrupted (∆ cyabrB2) mutant by knockout of the glgC (slr1176) gene encoding glucose-1-phosphate adenylyltransferase. However, complete knockout could not be attained, suggesting that accumulation of glycogen is essential for the Δ cyabrB2 mutant. In this study, we introduced the cyabrB2 gene fused to the copper-inducible petE promoter into the ∆ cyabrB2 mutant. After complete knockout of glgC in the presence of copper, expression of P petE- cyabrB2 was turned off by copper removal to examine the effect of the double knockout of cyabrB2 and glgC. Metabolome analysis and electron microscopic observation revealed that the double knockout causes a large decrease of sugar phosphates in glycolytic and oxidative pentose phosphate pathways and an increase of organic acids in the tricarboxylic acid cycle, amino acids and storage compounds such as polyhydroxybutyrate. When the ability of production of free fatty acids was conferred, synergetic positive effects of knockout of cyabrB2 and glgC on productivity were observed by removal of both copper and nitrogen. The P petE- cyabrB2Δ glgC strain will further serve as a platform for studies on carbon allocation and metabolic engineering.

Inositol Hexakis Phosphate is the Seasonal Phosphorus Reservoir in the Deciduous Woody Plant Populus alba L.

Seasonal recycling of nutrients is an important strategy for deciduous perennials. Deciduous perennials maintain and expand their nutrient pools by the autumn nutrient remobilization and the subsequent winter storage throughout their long life. Phosphorus (P), one of the most important elements in living organisms, is remobilized from senescing leaves during autumn in deciduous trees. However, it remains unknown how phosphate is stored over winter. Here we show that in poplar trees (Populus alba L.), organic phosphates are accumulated in twigs from late summer to winter, and that IP6 (myo-inositol-1,2,3,4,5,6-hexakis phosphate: phytic acid) is the primary storage form. IP6 was found in high concentrations in twigs during winter and quickly decreased in early spring. In parenchyma cells of winter twigs, P was associated with electron-dense structures, similar to globoids found in seeds of higher plants. Various other deciduous trees were also found to accumulate IP6 in twigs during winter. We conclude that IP6 is the primary storage form of P in poplar trees during winter, and that it may be a common strategy for seasonal P storage in deciduous woody plants.

Effects of tiotropium on lung function in current smokers and never smokers with bronchial asthma.

The effects of tiotropium, an inhaled long-acting muscarinic antagonist, on lung function were investigated in current smokers and nonsmokers with asthma treated with inhaled corticosteroids (ICSs) and other asthma controllers: inhaled long-acting ß2 agonists, leukotriene receptor antagonists, and/or theophylline. We conducted a double-blind, placebo-controlled study of an inhaled single dose of tiotropium in 9 asthmatics currently smoking and 9 asthmatics who have never smoked in a crossover manner. Lung function was measured before and 1, 3, and 24 h after inhalation of 18 µg of tiotropium or a placebo. The primary outcome was a change in forced expiratory volume in 1 s (FEV1) from the baseline, and the secondary outcomes were changes in peak expiratory flow rate (PEFR), V˙50, and V˙25. At baseline, asthmatics with and without a smoking history had a mean FEV1 of 2590 ml and 2220 ml and were taking a mean dose of ICSs of 1208 and 1000 µg/day, respectively. The increase from the baseline FEV1 was 169 ml and 105 ml higher at 3 h after tiotropium than after the placebo in current smokers and nonsmokers, respectively. PEFR, V˙50, and V˙25 were also significantly increased after tiotropium as compared with the placebo in both study groups. Changes in FEV1 and PEFR tended to be greater in asthmatics currently smoking than in subjects who have never smoked, although there were no statistical differences at any time points. Tiotropium resulted in improved lung function and symptoms both in current smoker and nonsmoker asthmatics. These findings suggest that tiotropium will provide a new strategy for the treatment of bronchial asthma.

Buoyancy Limitation of Filamentous Cyanobacteria under Prolonged Pressure due to the Gas Vesicles Collapse.

Freshwater cyanobacterium Pseudanabaena galeata were cultured in chambers under artificially generated pressures, which correspond to the hydrostatic pressures at deep water. Variations occurred in gas vesicles volume, and buoyancy state of cells under those conditions were analyzed at different time intervals (5 min, 1 day, and 5 days). Variations in gas vesicles morphology of cells were observed by transmission electron microscopy images. Settling velocity (Vs) of cells which governs the buoyancy was observed with the aid of a modified optical microscope. Moreover, effects of the prolonged pressure on cell ballast composition (protein and polysaccharides) were examined. Elevated pressure conditions reduced the cell ballast and caused a complete disappearance of gas vesicles in Pseudanabaena galeata cells. Hence cyanobacteria cells were not able to float within the study period. Observations and findings of the study indicate the potential application of hydrostatic pressure, which naturally occurred in hypolimnion of lakes, to inhibit the re-suspension of cyanobacteria cells.

Prevalence of Airflow Limitation Defined by Pre- and Post-Bronchodilator Spirometry in a Community-Based Health Checkup: The Hisayama Study.

Spirometry in health checkup may contribute to early diagnosis of chronic obstructive pulmonary disease (COPD) and asthma. Although post-bronchodilator airflow limitation is essential for definite diagnosis of COPD and post-bronchodilator normalization of airflow is suggestive of asthma, this test has not been prevailed in health checkup. The objective of this study was to estimate the prevalence of airflow limitation defined by pre- and post-bronchodilator spirometry in health checkup. Post-bronchodilator spirometry was conducted for participants with airflow limitation in a town-wide health checkup for residents aged 40 years and older in Hisayama, a town in the western part of Japan. The prevalence of pre- and post-bronchodilator airway limitation defined by FEV1/FVC < 70% were estimated. A total of 2,232 participants underwent pre-bronchodilator spirometry. In males, the age of current smokers was significantly younger than those of never smokers and former smokers. In females, the ages of current- and former smokers were significantly younger than never smokers. The values of %FEV1 and %FVC in current smokers were significantly lower than those in former smokers and never smokers. Two hundred sixty nine subjects, 85% of total subjects with a pre-bronchodilator FEV1/FVC < 70%, completed post-bronchodilator spirometry. The prevalence of pre-bronchodilator airflow limitation was 14.6% in males and 13.7% in females, and the prevalence of post-bronchodilator airway limitation was 8.7% and 8.7%, respectively. Post-bronchodilator spirometry in health checkup would reduce the number of subjects with probable COPD to two-third. Recommendation for those examinees to take further evaluations may pave the way for early intervention.

Photoassimilation, assimilate translocation and plasmodesmal biogenesis in the source leaves of Arabidopsis thaliana grown under an increased atmospheric CO2 concentration.

Using 18-day-old Arabidopsis thaliana seedlings grown under increased (780 p.p.m., experimental plants) or ambient (390 p.p.m., control plants) CO2 conditions, we evaluated (14)CO2 photoassimilation in and translocation from representative source leaves. The total (14)CO2 photoassimilation amounts increased in the third leaves of the experimental plants in comparison with that found for the third leaves of the control plants, but the rates were comparable for the first leaves of the two groups. In contrast, translocation of labeled assimilates doubled in the first leaves of the experimental group, whereas translocation was, at best, passively enhanced even though photoassimilation increased in their third leaves. The transcript levels of the companion cell-specific sucrose:H(+) symporter gene SUC2 were not significantly affected in the two groups of plants, whereas those of the sucrose effluxer gene SWEET12 and the sieve element-targeted sucrose:H(+) symporter gene SUT4 were up-regulated in the experimental plants, suggesting up-regulation of SUT4-dependent apoplastic phloem loading. Compared with SUC2, SUT4 is a minor component that is expressed in companion cells but functions in sieve elements after transfer through plasmodesmata. The number of aniline blue-stained spots for plasmodesma-associated callose in the midrib wall increased in the first leaf of the experimental plants but was comparable in the third leaf between the experimental and control plants. These results suggest that A. thaliana responds to greater than normal concentrations of CO2 differentially in the first and third leaves in regards to photoassimilation, assimilate translocation and plasmodesmal biogenesis.

Deletion of the transcriptional regulator cyAbrB2 deregulates primary carbon metabolism in Synechocystis sp. PCC 6803.

cyAbrB is a transcriptional regulator unique to and highly conserved among cyanobacterial species. A gene-disrupted mutant of cyabrB2 (sll0822) in Synechocystis sp. PCC 6803 exhibited severe growth inhibition and abnormal accumulation of glycogen granules within cells under photomixotrophic conditions. Within 6 h after the shift to photomixotrophic conditions, sodium bicarbonate-dependent oxygen evolution activity markedly declined in the ΔcyabrB2 mutant, but the decrease in methyl viologen-dependent electron transport activity was much smaller, indicating inhibition in carbon dioxide fixation. Decreases in the transcript levels of several genes related to sugar catabolism, carbon dioxide fixation, and nitrogen metabolism were also observed within 6 h. Metabolome analysis by capillary electrophoresis mass spectrometry revealed that several metabolites accumulated differently in the wild-type and mutant strains. For example, the amounts of pyruvate and 2-oxoglutarate (2OG) were significantly lower in the mutant than in the wild type, irrespective of trophic conditions. The growth rate of the ΔcyabrB2 mutant was restored to a level comparable to that under photoautotrophic conditions by addition of 2OG to the growth medium under photomixotrophic conditions. Activities of various metabolic processes, including carbon dioxide fixation, respiration, and nitrogen assimilation, seemed to be enhanced by 2OG addition. These observations suggest that cyAbrB2 is essential for the active transcription of genes related to carbon and nitrogen metabolism upon a shift to photomixotrophic conditions. Deletion of cyAbrB2 is likely to deregulate the partition of carbon between storage forms and soluble forms used for biosynthetic purposes. This disorder may cause inactivation of cellular metabolism, excess accumulation of reducing equivalents, and subsequent loss of viability under photomixotrophic conditions.

Localization of Lipid Droplets in Embryonic Axis Radicle Cells of Soybean Seeds under Various Imbibition Regimes Indicates Their Role in Desiccation Tolerance.

Desiccation tolerance allows plant seeds to remain viable during desiccation and subsequent re-hydration. In this study, we tried to develop an experimental system to understand the difference between desiccation tolerant and desiccation sensitive radicle cells by examining excised embryonic axes after re-desiccation and subsequent imbibition under various regimes. Embryonic axes excised from soybean (Glycine max (L.) Merr.) seeds imbibed for 3 h to 15 h which remained attached to the cotyledons during imbibition would grow normally after 24 h of desiccation and re-imbibition on wet filter paper. By contrast, when the embryonic axes excised after 3 h imbibition of seeds were kept on wet filter paper for 12 h to 16 h, their growth was significantly retarded after 24 h of desiccation and subsequent re-imbibition. Numerous lipid droplets were observed lining the plasma membrane and tonoplasts in radicle cells of desiccation tolerant embryonic axes before and after desiccation treatment. By contrast, the lipid droplets lining the plasma membrane and tonoplasts became very sparse in radicle cells that were placed for longer times on wet filter paper before desiccation. We observed a clear correlation between the amount of lipid droplets lining plasma membranes and the ability to grow after desiccation and re-imbibition of the excised embryonic axes. In addition to the reduction of lipid droplets in the cells, a gradual increase in starch grains was observed. Large starch grains accumulated in the radicle cells of those axes that failed to grow further.

Using a novel CLEM system to localize lipid droplets and membranes in desiccated embryonic axis cells of soybean seeds.

Lipid droplets and membranes in radicle cells from desiccated embryonic axes of soybean (Glycine max) seeds were examined by a recently developed correlative light and electron microscopy system, which has been designed to facilitate the observation of identical locations using an upright reflected light microscope and compact SEM successively with minimum time lapse. Lipids are major components of membranes and are also stored in numerous lipid droplets lining plasma membranes in many seed cells. Fluorescently stained lipid droplets and membranes in the desiccated radicle cells were mainly located along the surface of shrunk protoplasm and around presumptive protein bodies, which will turn into vacuoles and increase their volume for radicle protrusion. Co-localization of lipid droplets and membranes suggests the presence of a membrane protection mechanism during desiccation and rehydration processes that ensures prompt elongation of radicle cells during germination.

Acylated plastoquinone is a novel neutral lipid accumulated in cyanobacteria.

Although cyanobacteria do not possess bacterial triacylglycerol (TAG)-synthesizing enzymes, the accumulation of TAGs and/or lipid droplets has been repeatedly reported in a wide range of species. In most cases, the identification of TAG has been based on the detection of the spot showing the mobility similar to the TAG standard in thin-layer chromatography (TLC) of neutral lipids. In this study, we identified monoacyl plastoquinol (acyl PQH) as the predominant molecular species in the TAG-like spot from the unicellular Synechocystis sp. PCC 6803 (S.6803) as well as the filamentous Nostocales sp., Nostoc punctiforme PCC 73102, and Anabaena sp. PCC 7120. In S.6803, the accumulation level of acyl PQH but not TAG was affected by deletion or overexpression of slr2103, indicating that acyl PQH is the physiological product of Slr2103 having homology with the eukaryotic diacylglycerol acyltransferase-2 (DGAT2). Electron microscopy revealed that cyanobacterial strains used in this study do not accumulate lipid droplet structures such as those observed in oleaginous microorganisms. Instead, they accumulate polyhydroxybutyrate (PHB) granules and/or aggregates of alkane, free C16 and C18 saturated fatty acids, and low amounts of TAG in the cytoplasmic area, which can be detected by staining with a fluorescent dye specific to neutral lipids. Unlike these lipophilic materials, acyl PQH is exclusively localized in the membrane fraction. There must be DGAT2-like enzymatic activity esterifying de novo-synthesized C16 and C18 fatty acids to PQH2 in the thylakoid membranes.

A possible involvement of autophagy in amyloplast degradation in columella cells during hydrotropic response of Arabidopsis roots.

Seedling roots display not only gravitropism but also hydrotropism, and the two tropisms interfere with one another. In Arabidopsis (Arabidopsis thaliana) roots, amyloplasts in columella cells are rapidly degraded during the hydrotropic response. Degradation of amyloplasts involved in gravisensing enhances the hydrotropic response by reducing the gravitropic response. However, the mechanism by which amyloplasts are degraded in hydrotropically responding roots remains unknown. In this study, the mechanistic aspects of the degradation of amyloplasts in columella cells during hydrotropic response were investigated by analyzing organellar morphology, cell polarity and changes in gene expression. The results showed that hydrotropic stimulation or systemic water stress caused dramatic changes in organellar form and positioning in columella cells. Specifically, the columella cells of hydrotropically responding or water-stressed roots lost polarity in the distribution of the endoplasmic reticulum (ER), and showed accelerated vacuolization and nuclear movement. Analysis of ER-localized GFP showed that ER redistributed around the developed vacuoles. Cells often showed decomposing amyloplasts in autophagosome-like structures. Both hydrotropic stimulation and water stress upregulated the expression of AtATG18a, which is required for autophagosome formation. Furthermore, analysis with GFP-AtATG8a revealed that both hydrotropic stimulation and water stress induced the formation of autophagosomes in the columella cells. In addition, expression of plastid marker, pt-GFP, in the columella cells dramatically decreased in response to both hydrotropic stimulation and water stress, but its decrease was much less in the autophagy mutant atg5. These results suggest that hydrotropic stimulation confers water stress in the roots, which triggers an autophagic response responsible for the degradation of amyloplasts in columella cells of Arabidopsis roots.

Expression and localization of two SecA homologs in the unicellular red alga Cyanidioschyzon merolae.

SecA is an ATP-driven motor for Sec translocase that participates in bacterial protein export and thylakoidal import in plants. We have reported that Cyanidioschyzon merolae, a unicellular red alga, possesses a nuclear-encoded secA(nuc) and a plastid-encoded secA(pt) gene. In this study we found that the amount of SecA(nuc) protein almost quadrupled at high temperature, whereas that of the SecA(pt) protein increased far less. We were also able to determine the localization of both SecAs to the chloroplast by immunofluorescence and immunoelectron microscopy. We suggest that SecA(nuc) has an important role in the chloroplast at high temperatures.

Physiological roles of the cyAbrB transcriptional regulator pair Sll0822 and Sll0359 in Synechocystis sp. strain PCC 6803.

All known cyanobacterial genomes possess multiple copies of genes encoding AbrB-like transcriptional regulators, known as cyAbrBs, which are distinct from those conserved among other bacterial species. In this study, we addressed the physiological roles of Sll0822 and Sll0359, the two cyAbrBs in Synechocystis sp. strain PCC 6803, under nonstress conditions (20 µmol of photons m⁻² s⁻¹ in ambient CO2). When the sll0822 gene was disrupted, the expression levels of nitrogen-related genes such as urtA, amt1, and glnB significantly decreased compared with those in the wild-type cells. Possibly due to the increase of the cellular carbon/nitrogen ratio in the sll0822-disrupted cells, a decrease in pigment contents, downregulation of carbon-uptake related genes, and aberrant accumulation of glycogen took place. Moreover, the mutant exhibited the decrease in the expression level of cytokinesis-related genes such as ftsZ and ftsQ, resulting in the defect in cell division and significant increase in cell size. The pleiotrophic phenotype of the mutant was efficiently suppressed by the introduction of Sll0822 and also partially suppressed by the introduction of Sll0359. When His-tagged cyAbrBs were purified from overexpression strains, Sll0359 and Sll0822 were copurified with each other. The cyAbrBs in Synechocystis sp. strain PCC 6803 seem to interact with each other and regulate carbon and nitrogen metabolism as well as the cell division process under nonstress conditions.

Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls.

The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.

TEM observation of compacted DNA of Synechococcus elongatus PCC 7942 using DRAQ5 labeling with DAB photooxidation and osmium black.

To visualize the fine structure of compacted DNA of Synechococcus elongatus PCC 7942, which appears at a specific time in the regular light/dark cycle prior to cell division, ChromEM with some modifications was applied. After staining DNA with DRAQ5, the cells were fixed and irradiated by red laser in the presence of 3,3'-diaminobenzidine and subsequently fixed with OsO4. A system with He-Ne laser (633 nm) was set up for efficient irradiation of the bacterial cells in aqueous solution. The compacted DNA was visualized by transmission electron microscopy, in ultrathin sections as electron dense staining by osmium black.

CccS and CccP are involved in construction of cell surface components in the cyanobacterium Synechocystis sp. strain PCC 6803.

We have previously identified two target genes (slr1667 and slr1668) for transcriptional regulation by a cAMP receptor protein, SYCRP1, in a cAMP-dependent manner. For this study we investigated the localizations of products of slr1667 and slr1668 (designated cccS and cccP, respectively) biochemically and immunocytochemically, and examined the phenotypes of their disruptants. CccS protein was detected in the culture medium and the acid-soluble fraction containing proteins derived from outside the outer membrane. Disruptants of cccS and cccP showed a more or less similar pleiotropic phenotype. Several proteins secreted into the culture medium or retained on the outside of the outer membrane were greatly reduced in both disruptants compared with the wild type. Electron microscopy revealed that the cccS disruptant lacked the thick pili responsible for motility and that the cccP disruptant had almost no discernible thick pili on its cell surface. Both disruptants largely secreted far greater amounts of yellow pigments into the culture medium than did the wild type. Furthermore, the disruptions reduced the amount of UV-absorbing compound(s) extractable from the exopolysaccharide layer. These results suggest that the cccS and cccP genes are involved in the construction of cell surface components in Synechocystis sp. strain PCC 6803.

Rice BRITTLE CULM 3 (BC3) encodes a classical dynamin OsDRP2B essential for proper secondary cell wall synthesis.

"Brittle culm" mutants found in Gramineae crops are suitable materials to study the mechanism of secondary cell wall formation. Through positional cloning, we have identified a gene responsible for the brittle culm phenotype in rice, brittle culm 3 (bc3). BC3 encodes a member of the classical dynamin protein family, a family known to function widely in membrane dynamics. The bc3 mutation resulted in reductions of 28-36% in cellulose contents in culms, leaves, and roots, while other cell wall components remained unaffected. Reductions of cell wall thickness and birefringence were observed in both fiber (sclerenchyma) and parenchymal cells, together with blurring of the wall's layered structures. From promoter-GUS analyses, it was suggested that BC3 expression is directly correlated with active secondary cell wall synthesis. These results suggest that BC3 is tightly involved in the synthesis of cellulose and is essential for proper secondary cell wall construction.