Effect of light fluctuations on photosynthesis and metabolic flux in Synechocystis sp. PCC 6803.

In nature, photosynthetic organisms are exposed to fluctuating light, and their physiological systems must adapt to this fluctuation. To maintain homeostasis, these organisms have a light fluctuation photoprotective mechanism, which functions in both photosystems and metabolism. Although the photoprotective mechanisms functioning in the photosystem have been studied, it is unclear how metabolism responds to light fluctuations within a few seconds. In the present study, we investigated the metabolic response of Synechocystis sp. PCC 6803 to light fluctuations using 13 C-metabolic flux analysis. The light intensity and duty ratio were adjusted such that the total number of photons or the light intensity during the low-light phase was equal. Light fluctuations affected cell growth and photosynthetic activity under the experimental conditions. However, metabolic flux distributions and cofactor production rates were not affected by the light fluctuations. Furthermore, the estimated ATP and NADPH production rates in the photosystems suggest that NADPH-consuming electron dissipation occurs under fluctuating light conditions. Although we focused on the water-water cycle as the electron dissipation path, no growth effect was observed in an flv3-disrupted strain under fluctuating light, suggesting that another path contributes to electron dissipation under these conditions.

Recent advances in lignocellulosic biomass white biotechnology for bioplastics.

Lignocellulosic biomass has great potential as an inedible feedstock for bioplastic synthesis, although its use is still limited compared to current edible feedstocks of glucose and starch. This review focuses on recent advances in the production of biopolymers and biomonomers from lignocellulosic feedstocks with downstream processing and chemical polymer syntheses. In microbial production, four routes composed of existing poly (lactic acid) and polyhydroxyalkanoates (PHAs) and the emerging biomonomers of itaconic acid and aromatic compounds were presented to review present challenges and future perspectives, focusing on the use of lignocellulosic feedstocks. Recently, advances in purification technologies decreased the number of processes and their environmental burden. Additionally, the unique structures and high-performance of emerging lignocellulose-based bioplastics have expanded the possibilities for the use of bioplastics. The sequence of processes provides insight into the emerging technologies that are needed for the practical use of bioplastics made from lignocellulosic biomass.

Proteome analysis of response to different spectral light irradiation in Synechocystis sp. PCC 6803.

In cyanobacteria, it is known that the excitation ratios of photosystem (PS) I and PSII changes with the wavelength of irradiated light due to mobile phycobilisome (PBS) and spillover, affecting the photosynthetic ATP/NADPH synthesis ratio and metabolic flux state. However, the mechanisms by which these changes are controlled have not been well studied. In this study, we performed a targeted proteomic analysis of Synechocystis sp. PCC 6803 under different spectral light conditions to clarify the regulation mechanisms of mobile PBS, spillover and metabolisms under different light qualities at the protein level. The results showed an increase in the amount of proteins mainly involved in CO2 fixation under Red1 light conditions with a high specific growth rate, suggesting that the rate of intracellular metabolism is controlled by the rate of carbon uptake, not by changes in the amount of each enzyme. Correlation analysis between protein levels and PSI/PSII excitation ratios revealed that PsbQUY showed high correlations and significantly increased under Blue and Red2 light conditions, where the PSI/PSII excitation ratio was higher due to spillover. In the strains lacking the genes encoding these proteins, a decrease in the PSI/PSII excitation ratio was observed, suggesting that PsbQUY contribute to spillover occurrence. SIGNIFICANCE: In cyanobacteria, the photosynthetic apparatus's responses, such as state transition [mobile PBS and spillover], occur due to the intensity and wavelength of irradiated light, resulting in changes in photosynthetic electron transport and metabolic flux states. Previous studies have analyzed the response of Synechocystis sp. PCC 6803 to light intensity from various directions, but only spectroscopic analysis of the photosynthetic apparatus has been done on the response to changes in the wavelength of irradiated light. This study analyzed the response mechanisms of mobile PBS, spillover, photosynthetic, and metabolic systems in Synechocystis sp. PCC 6803 under six different spectral light conditions by a targeted proteomic analysis. As a result, many proteins were successfully quantified, and the metabolic enzymes and photosynthetic apparatus were analyzed using an integrated approach. Principal component and correlation analyses and volcano plots revealed that the PSII subunits PsbQ, PsbU, and PsbY have a strong correlation with the PSI/PSII excitation ratio and contribute to spillover occurrence. Thus, statistical analysis based on proteome data revealed that PsbQ, PsbU, and PsbY are involved in spillover, as revealed by spectroscopic analysis.

Dynamism of Metabolic Carbon Flow of Starch and Lipids in Chlamydomonas debaryana.

Microalgae have the potential to recycle CO2 as starch and triacylglycerol (TAG), which provide alternative source of biofuel and high added-value chemicals. Starch accumulates in the chloroplast, whereas TAG accumulates in the cytoplasmic lipid droplets (LD). Preferential accumulation of starch or TAG may be achieved by switching intracellular metabolic carbon flow, but our knowledge on this control remains limited. Are these two products mutually exclusive? Or, does starch act as a precursor to TAG synthesis, or vice versa? To answer these questions, we analyzed carbon flow in starch and lipids using a stable isotope 13C in Chlamydomonas debaryana NIES-2212, which accumulates, without nutrient limitation, starch in the exponential growth phase and TAG in the stationary phase. Pulse labeling experiments as well as pulse labeling and chase experiments were conducted, and then, gas chromatography-mass spectrometry (GC-MS) analysis was performed on starch-derived glucose and lipid-bound fatty acids. We exploited the previously developed method of isotopomer analysis to estimate the proportion of various pools with different isotopic abundance. Starch turned over rapidly to provide carbon for the synthesis of fatty acids in the exponential phase cells. Most fatty acids showed rapid and slow components of metabolism, whereas oleic acid decayed according to a single exponential curve. Highly labeled population of fatty acids that accumulated during the initial labeling decreased rapidly, and replaced by low abundance population during the chase time, indicating that highly labeled fatty acids were degraded and the resulting carbons were re-used in the re-synthesis with about 9-fold unlabeled, newly fixed carbons. Elongation of C16-C18 acids in vivo was indicated by partially labeled C18 acids. The accumulation of TAG in the stationary growth phase was accounted for by both de novo synthesis and remodeling of membrane lipids. These results suggest that de novo synthesis of starch and TAG was rapid and transient, and also almost independent to each other, but there is a pool of starch quickly turning over for the synthesis of fatty acids. Fatty acids were also subject to re-synthesis. Evidence was also provided for remodeling of lipids, namely, re-use of acyl groups in polar lipids for TAG synthesis.

Thioredoxin pathway in Anabaena sp. PCC 7120: activity of NADPH-thioredoxin reductase C.

To understand the physiological role of NADPH-thioredoxin reductase C (NTRC) in cyanobacteria, we investigated an NTRC-deficient mutant strain of Anabaena sp., PCC 7120, cultivated under different regimes of nitrogen supplementation and light exposure. The deletion of ntrC did not induce a change in the cell structure and metabolic pathways. However, time-dependent changes in the abundance of specific proteins and metabolites were observed. A decrease in chlorophyll a was correlated with a decrease in chlorophyll a biosynthesis enzymes and photosystem I subunits. The deletion of ntrC led to a deregulation of nitrogen metabolism, including the NtcA accumulation and heterocyst-specific proteins while nitrate ions were available in the culture medium. Interestingly, this deletion resulted in a redox imbalance, indicated by higher peroxide levels, higher catalase activity and the induction of chaperones such as MsrA. Surprisingly, the antioxidant protein 2-CysPrx was downregulated. The deficiency in ntrC also resulted in the accumulation of metabolites such as 6-phosphogluconate, ADP and ATP. Higher levels of NADP+ and NADPH partly correlated with higher G6PDH activity. Rather than impacting protein expression levels, NTRC appears to be involved in the direct regulation of enzymes, especially during the dark-to-light transition period.

Role of type I NADH dehydrogenase in Synechocystis sp. PCC 6803 under phycobilisome excited red light.

Cyanobacterial type I NADH dehydrogenase (NDH-1) is involved in various bioenergetic reactions including respiration, cyclic electron transport (CET), and CO2 uptake. The role of NDH-1 is usually minor under normal growth conditions and becomes important under stress conditions. However, in our previous study, flux balance analysis (FBA) simulation predicted that the drive of NDH-1 as CET pathway with a photosystem (PS) I/PSII excitation ratio around 1.0 contributes to achieving an optimal specific growth rate. In this study, to experimentally elucidate the predicted functions of NDH-1, first, we measured the PSI/PSII excitation ratios of Synechocystis sp. PCC 6803 grown under four types of spectral light conditions. The specific growth rate was the highest and PSI/PSII excitation ratio was with 0.88 under the single-peak light at 630 nm (Red1). Considering this measured excitation ratios, FBA simulation predicted that NDH-1-dependent electron transport was the major pathway under Red1. Moreover, in actual culture, an NDH-1 deletion strain had slower growth rate than that of wild type only under Red1 light condition. Therefore, we experimentally demonstrated that NDH-1 plays an important role under optimal light conditions such as Red1 light, where Synechocystis exhibits the highest specific growth rate and PSI/PSII excitation ratio of around 1.0.

Estimation of linear and cyclic electron flows in photosynthesis based on 13C-metabolic flux analysis.

Photosynthetic organisms produce ATP and NADPH using light as an energy source and further utilize these cofactors during metabolism. Photosynthesis involves linear and cyclic electron flows; as the cyclic electron flow produces ATP more effectively than the linear electron flow without NADPH, the cell efficiently adjusts ATP and NADPH production using the two different pathways. Nevertheless, direct measurement of ATP and NADPH production during photosynthesis has been difficult. In the present study, the photosynthetic ATP and NADPH production rates of Synechocystis sp. PCC 6803 under three different single peak wavelength lights (blue: 470 nm, R630: 630 nm, and R680: 680 nm) were evaluated based on 13C-metabolic flux analysis (13C-MFA) by considering the mass balance of ATP and NADPH between photosynthesis and metabolism. The ratios of ATP/NADPH production via photosynthesis were estimated as 3.13, 1.70, and 2.10 under blue, R630, and R680 light conditions, respectively. Moreover, the linear and cyclic electron flow ratios were estimated to be 1.1-2.2, 0.2-0.5, and 0.5-1.0 under blue, R630, and R680 light conditions, respectively. The predicted linear and cyclic electron flow ratios were consistent with the excitation ratio between photosystems I and II, as observed in the steady-state fluorescence spectra.

Assessment of Protein Content and Phosphorylation Level in Synechocystis sp. PCC 6803 under Various Growth Conditions Using Quantitative Phosphoproteomic Analysis.

The photosynthetic apparatus and metabolic enzymes of cyanobacteria are subject to various controls, such as transcriptional regulation and post-translational modifications, to ensure that the entire cellular system functions optimally. In particular, phosphorylation plays key roles in many cellular controls such as enzyme activity, signal transduction, and photosynthetic apparatus restructuring. Therefore, elucidating the governing functions of phosphorylation is crucial to understanding the regulatory mechanisms underlying metabolism and photosynthesis. In this study, we determined protein content and phosphorylation levels to reveal the regulation of intracellular metabolism and photosynthesis in Synechocystis sp. PCC 6803; for this, we obtained quantitative data of proteins and their phosphorylated forms involved in photosynthesis and metabolism under various growth conditions (photoautotrophic, mixotrophic, heterotrophic, dark, and nitrogen-deprived conditions) using targeted proteomic and phosphoproteomic analyses with nano-liquid chromatography-triple quadrupole mass spectrometry. The results indicated that in addition to the regulation of protein expression, the regulation of phosphorylation levels of cyanobacterial photosynthetic apparatus and metabolic enzymes was pivotal for adapting to changing environmental conditions. Furthermore, reduced protein levels of CpcC and altered phosphorylation levels of CpcB, ApcA, OCP, and PsbV contributed to the cellular response of the photosynthesis apparatus to nitrogen deficiency.

Formation of Spherical Palmelloid Colony with Enhanced Lipid Accumulation by Gel Encapsulation of Chlamydomonas debaryana NIES-2212.

Microalgae such as Chlamydomonas reinhardtii accumulate triacylglycerol (TAG), which is a potential source of biofuels, under stress conditions such as nitrogen deprivation, whereas Chlamydomonas debaryana NIES-2212 has previously been identified and characterized as one of the rare species of Chlamydomonas, which massively accumulates TAG in the stationary phase without external stress. As the high density of the cells in the stationary phase was supposed to act as a trigger for the accumulation of TAG in C. debaryana, in this study, C. debaryana was encapsulated in a Ca2+-alginate gel for the culture with high cell density. We discovered that the growth of the encapsulated cells resulted in the formation of spherical palmelloid colonies with high cell density, where daughter cells with truncated flagella remained wrapped within the mother cell walls. Interestingly, gel encapsulation markedly promoted proliferation of C. debaryana cells, and the encapsulated cells reached the stationary phase earlier than that of the free-living cells. Gel encapsulation also enhanced TAG accumulation. Gene expression analysis revealed that two genes of acyltransferases, DGAT1 and DGTT3, were upregulated in the stationary phase of free-living C. debaryana. In addition, the gene expression of these acyltransferases increased earlier in the encapsulated cells than that in the free-living cells. The enhanced production of TAG by alginate gel encapsulation was not found in C. reinhardtii which is known to use a different repertoire of acyltransferases in lipid accumulation.

Flux balance analysis of cyanobacteria reveals selective use of photosynthetic electron transport components under different spectral light conditions.

Cyanobacteria acclimate and adapt to changing light conditions by controlling the energy transfer between photosystem I (PSI) and II (PSII) and pigment composition. Photosynthesis is driven by balancing the excitation between PSI and PSII. To predict the detailed electron transfer flux of cyanobacteria, we refined the photosynthesis-related reactions in our previously reconstructed genome-scale model. Two photosynthetic bacteria, Arthrospira and Synechocystis, were used as models. They were grown under various spectral light conditions and flux balance analysis (FBA) was performed using photon uptake fluxes into PSI and PSII, which were converted from each light spectrum by considering the photoacclimation of pigments and the distribution ratio of phycobilisome to PSI and PSII. In Arthrospira, the FBA was verified with experimental data using six types of light-emitting diodes (White, Blue, Green, Yellow, Red1, and Red2). FBA predicted the cell growth of Synechocystis for the LEDs, excepting Red2. In an FBA simulation, cells used respiratory terminal oxidases and two NADH dehydrogenases (NDH-1 and NDH-2) to balance the PSI and PSII excitations depending on the light conditions. FBA simulation with our refined model functionally implicated NDH-1 and NDH-2 as a component of cyclic electron transport in the varied light environments.

Time-resolved analysis of short term metabolic adaptation at dark transition in Synechocystis sp. PCC 6803.

In photosynthetic organisms, such as cyanobacteria, ATP and NADPH are generated through the light reaction, and then are used for CO2 fixation in the dark reaction. As light intensity always fluctuates under natural conditions, balancing the cofactor regeneration and consumption is essential to maintain active CO2 fixation as well as for metabolic engineering of strains that produce biochemicals. In this study, a time-resolved metabolome analysis of Synechocystis sp. PCC 6803 (PCC6803) was conducted to investigate a metabolic adaptation at 0-15 min after a sudden shift from light to dark conditions. Rapid accumulation of sedoheptulose 7-phosphate, ribulose 5-phosphate, xylulose 5-phosphate, and 6-phosphogluconate suggested that the central metabolism of PCC6803 was regulated by inactivation of phosphoribulokinase and activation of glucose-6-phosphate dehydrogenase (G6PDH) probably via the redox regulation. The culture and metabolic profile of the Δzwf strain lacking G6PDH showed that the role of G6PDH in regeneration of NADPH could be complemented by the activation of isocitrate dehydrogenase in the TCA cycle, indicating the importance of the rapid regulation of NADPH regeneration after the shift to dark conditions. The mechanism underlying metabolic regulation is also useful for metabolic engineering of PCC6803, as the Δzwf strain produced higher amount of organic acids than wild type.

Targeted proteome analysis of microalgae under high-light conditions by optimized protein extraction of photosynthetic organisms.

Cell disruption and protein solubilization protocols for the relative quantification of individual subunits in photosystems were developed for photosynthetic organisms including cyanobacterium Synechocystis sp. PCC 6803, green-algae Chlamydomonas reinhardtii, and seed plant Arabidopsis thaliana. The optimal methods for the disruption of Chlamydomonas, Synechocystis, and Arabidopsis cells were sonication, microbeads (Φ approximately 0.1 mm), and large beads (Φ = 5.0 mm), respectively. Extraction of the total proteins exceeded 90% using each optimal cell disruption method. Solubilization efficiency of membrane proteins was improved by the phase transfer surfactant (PTS) method. Ninety seven and 114 proteins from Chlamydomonas and Synechocystis, respectively, including membrane proteins such as photosystem proteins, ATP synthase, and NADH dehydrogenase, were successfully analyzed by nano-liquid chromatography tandem mass spectrometry. These results also indicated the improved efficiency of solubilization and trypsin digestion using PTS buffer. The results of the relative quantitative evaluation of photosystem subunits in Chlamydomonas and Synechocystis grown under high-light conditions were consistent with those of previous studies. Thus, the optimal cell disruption and PTS methods allow for comprehensive relative quantitative proteome analysis of photosynthetic organisms. Additionally, NdhD1 and NdhF1, which are NDH-1 subunit homologs, were increased under high-light conditions, suggesting that the NDH-1L complex, including NdhD1 and NdhF1, is increased under high-light conditions. The relative quantitative proteome analysis of individual subunits indicates the diverse functions of NDH-1 protein.

Analysis and modeling of the inverted bioconvection in Chlamydomonas reinhardtii: emergence of plumes from the layer of accumulated cells.

Bioconvection is a convective flow found in a suspension of motile cells that swim against gravity, and is a primitive form of order formation of cells, which has been studied both experimentally and theoretically. We formulate here an inverted bioconvection occurring in a suspension of phototactic cells in a high-density medium, which is illuminated from the bottom. We used a highly phototactic strain 137c of Chlamydomonas reinhardtii in the experiments. Using a custom-made lateral microscope, we observed a close view of cellular dynamics in the initiation of inverted bioconvection. In conventional bioconvection, convective flows of cells are formed spontaneously with or without formation of the surface cell layer. In inverted convection, a crowded cell layer was initially formed at the bottom, which was a prerequisite for the subsequent emergence of plumes, namely, floating populations of cells. The plume formation was a result of neither uneven initial cell density nor unequal light intensity. Based on detailed analysis of individual cells, we constructed a model of inverted bioconvection, in which each cell experiences a transition between two modes of movement: phototactically swimming cell and non-motile cell aggregate. A simulation using the CompuCell3D software reproduced basic behaviors of the plume formation. The modal transition has not been a subject of basic studies, but provides an interesting target of study of cell-to-cell interactions.

Comparative Targeted Proteomics of the Central Metabolism and Photosystems in SigE Mutant Strains of Synechocystis sp. PCC 6803.

A targeted proteome analysis was conducted to investigate the SigE dependent-regulation of central metabolism in Synechocystis sp. PCC 6803 by directly comparing the protein abundance profiles among the wild type, a sigE deletion mutant (ΔsigE), and a sigE over-expression (sigEox) strains. Expression levels of 112 target proteins, including the central metabolism related-enzymes and the subunits of the photosystems, were determined by quantifying the tryptic peptides in the multiple reaction monitoring (MRM) mode of liquid-chromatography⁻triple quadrupole mass spectrometry (LC⁻MS/MS). Comparison with gene-expression data showed that although the abundance of Gnd protein was closely correlated with that of gnd mRNA, there were poor correlations for GdhA/gdhA and glycogen degradation-related genes such as GlgX/glgX and GlgP/glgP pairs. These results suggested that the regulation of protein translation and degradation played a role in regulating protein abundance. The protein abundance profile suggested that SigE overexpression reduced the proteins involved in photosynthesis and increased GdhA abundance, which is involved in the nitrogen assimilation pathway using NADPH. The results obtained in this study successfully demonstrated that targeted proteome analysis enables direct comparison of the abundance of central metabolism- and photosystem-related proteins.

Optimization of triacylglycerol and starch production in Chlamydomonas debaryana NIES-2212 with regard to light intensity and CO2 concentration.

Triacylglycerol (TAG) and starch produced by micro-algae are potential sources of biofuel. Our previous studies showed that the unicellular green alga, Chlamydomonas debaryana NIES-2212, which is a rare species of Chlamydomonas that possesses phosphatidylcholine (PC), is a seed organism for the development of biofuel producers. This alga accumulates large amounts of TAG and starch under completely photo-autotrophic conditions during stationary phase without nutrient deprivation. The present study was performed to optimize the growth conditions of this alga with regard to light intensity and CO2 concentration to improve the efficiency of TAG and starch production. The growth rate of C. debaryana was greater at higher light intensity, although there was no significant difference in the final cell density of the culture. The highest contents of TAG and starch, approximately 200 fmol cell-1 and 600 pg cell-1, respectively, were achieved with a light intensity of 200 µmol m-2 s-1 bubbled with air containing 5.0 % CO2. These results suggest that optimization of light intensity and CO2 concentration can enhance the productivity of TAG and starch by C. debaryana NIES-2212.

Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

Revisiting the Algal "Chloroplast Lipid Droplet": The Absence of an Entity That Is Unlikely to Exist.

The precise localization of the lipid droplets and the metabolic pathways associated with oil production are crucial to the engineering of microalgae for biofuel production. Several studies have reported detecting lipid droplets within the chloroplast of the microalga Chlamydomonas reinhardtii, which accumulates considerable amounts of triacylglycerol and starch within the cell under nitrogen deprivation or high-light stress conditions. Starch undoubtedly accumulates within the chloroplast, but there have been debates on the localization of the lipid droplets, which are cytosolic organelles in other organisms. Although it is impossible to prove an absence, we tried to repeat experiments that previously indicated the presence of lipid droplets in chloroplasts. Here, we present microscopic results showing no evidence for the presence of lipid droplets within the chloroplast stroma, even though some lipid droplets existed in close association with the chloroplast or were largely engulfed by the chloroplasts. These lipid droplets are cytosolic structures, distinct from the plastoglobules present in the chloroplast stroma. These results not only contrast with the old ideas but also point out that what were previously thought to be chloroplast lipid droplets are likely to be embedded within chloroplast invaginations in association with the outer envelope of the chloroplast without intervention of the endoplasmic reticulum. These findings point to the intriguing possibility of a tight metabolic flow from the chloroplast to the lipid droplet through a close association rather than direct contact of both organelles.

Single-Pixel Densitometry Revealed the Presence of Peptidoglycan in the Intermembrane Space of the Moss Chloroplast Envelope in Conventional Electron Micrographs.

Chloroplasts are believed to be descendants of ancestral cyanobacteria that have a peptidoglycan layer between the outer and the inner membranes. In particular, cyanelles having peptidoglycan in Cyanophora paradoxa are considered as evidence for the endosymbiotic origin of chloroplasts. The moss Physcomitrella patens has a complete set of genes involved in the synthesis of peptidoglycan, but a peptidoglycan layer has not been observed by conventional electron microscopy to date. Recently, a new metabolic labeling technique using a fluorescent probe was applied to visualize putative peptidoglycan surrounding the chloroplasts. The exact localization of the peptidoglycan, however, has not been clearly identified. Here we examined conventional electron micrographs of two types of moss materials (mutants or ampicillin-treated plants), one presumably having peptidoglycan and the other presumably lacking peptidoglycan, and analyzed in detail, by single-pixel densitometry, the electron density of the chloroplast envelope membranes and the intermembrane space. Statistical analysis showed that the relative electron density within the intermembrane space with respect to that of the envelope membranes was significantly higher in the materials presumably having peptidoglycan than in the materials presumably devoid of peptidoglycan. We consider this difference as bona fide evidence for the presence of peptidoglycan between the outer and the inner envelope membranes in the wild-type chloroplasts of the moss, although its density is lower than that in bacteria and cyanelles. We will also discuss this low-density peptidoglycan in the light of the phylogenetic origin of peptidoglycan biosynthesis enzymes.

Characterization of phosphoethanolamine-N-methyltransferases in green algae.

Phosphatidylcholine (PtdCho) is a common and abundant phospholipid in most eukaryotic organisms. Although it has been known that the model green alga Chlamydomonas reinhardtii lacks PtdCho, we recently detected PtdCho in four Chlamydomonas species. Homology search of draft genomic sequences of the four PtdCho-containing algae suggested existence of phosphoethanolamine-N-methyltransferase (PEAMT) in C. applanata and C. asymmetrica, which is the key enzyme in PtdCho biosynthesis in land plants. Here we analyzed the putative genes encoding PEAMT in C. applanata and C. asymmetrica, named CapPEAMT and CasPEAMT, respectively. In vitro assays with recombinant CapPEAMT and CasPEAMT indicated that they have the methylation activity for phosphoethanolamine, but not the methylation activity for phosphomonomethylethanolamine, in contrast with land plant PEAMTs, that possess the three successive methylation activities.

Lipid metabolism and potentials of biofuel and high added-value oil production in red algae.

Biomass production is currently explored in microalgae, macroalgae and land plants. Microalgal biofuel development has been performed mostly in green algae. In the Japanese tradition, macrophytic red algae such as Pyropia yezoensis and Gelidium crinale have been utilized as food and industrial materials. Researches on the utilization of unicellular red microalgae such as Cyanidioschyzon merolae and Porphyridium purpureum started only quite recently. Red algae have relatively large plastid genomes harboring more than 200 protein-coding genes that support the biosynthetic capacity of the plastid. Engineering the plastid genome is a unique potential of red microalgae. In addition, large-scale growth facilities of P. purpureum have been developed for industrial production of biofuels. C. merolae has been studied as a model alga for cell and molecular biological analyses with its completely determined genomes and transformation techniques. Its acidic and warm habitat makes it easy to grow this alga axenically in large scales. Its potential as a biofuel producer is recently documented under nitrogen-limited conditions. Metabolic pathways of the accumulation of starch and triacylglycerol and the enzymes involved therein are being elucidated. Engineering these regulatory mechanisms will open a possibility of exploiting the full capability of production of biofuel and high added-value oil. In the present review, we will describe the characteristics and potential of these algae as biotechnological seeds.