Yoshihiro Shiraiwa

Salt-Regulated Mannitol Metabolism in Algae

Mannitol, one of the most widely occurring sugar alcohol compounds, is found in bacteria, fungi, algae, and plants. In these organisms the compound acts as a compatible solute and has multiple functions, including osmoregulation, storage, and regeneration of reducing power, and scavenging of active oxygen species. Because of the diverse functions of mannitol, introducing the ability to accumulate it has been a hallmark of attempts to generate highly salt-tolerant transgenic plants. However, transgenic plants have not yet improved significantly in their salt tolerance. Recently, we purified and characterized 2 enzymes that biosynthesize mannitol, mannitol-1-phosphate dehydrogenase (M1PDH) and mannitol-1-phosphate-specific phosphatase, from the marine red alga Caloglossa continua, which grows in estuarine areas where tide levels fluctuate frequently. The activation of Caloglossa M1PDH is unique in that it is regulated by salt concentration at enzyme level. In this review we focus on the metabolism of mannitol, mainly in marine photosynthetic organisms, and suggest how this might be applied to producing salt-tolerant transgenic plants.

Historical perspective on microalgal and cyanobacterial acclimation to low- and extremely high-CO2 conditions

Reports in the 1970s from several laboratories revealed that the affinity of photosynthetic machinery for dissolved inorganic carbon (DIC) was greatly increased when unicellular green microalgae were transferred from high to low-CO2 conditions. This increase was due to the induction of carbonic anhydrase (CA) and the active transport of CO2 and/or HCO3− which increased the internal DIC concentration. The feature is referred to as the ‘CO2-concentrating mechanism (CCM)’. It was revealed that CA facilitates the supply of DIC from outside to inside the algal cells. It was also found that the active species of DIC absorbed by the algal cells and chloroplasts were CO2 and/or HCO3−, depending on the species. In the 1990s, gene technology started to throw light on the molecular aspects of CCM and identified the genes involved. The identification of the active HCO3− transporter, of the molecules functioning for the energization of cyanobacteria and of CAs with different cellular localizations in eukaryotes are examples of such successes. The first X-ray structural analysis of CA in a photosynthetic organism was carried out with a red alga. The results showed that the red alga possessed a homodimeric β-type of CA composed of two internally repeating structures. An increase in the CO2 concentration to several percent results in the loss of CCM and any further increase is often disadvantageous to cellular growth. It has recently been found that some microalgae and cyanobacteria can grow rapidly even under CO2 concentrations higher than 40%. Studies on the mechanism underlying the resistance to extremely high CO2 concentrations have indicated that only algae that can adopt the state transition in favor of PS I could adapt to and survive under such conditions. It was concluded that extra ATP produced by enhanced PS I cyclic electron flow is used as an energy source of H+-transport in extremely high-CO2 conditions. This same state transition has also been observed when high-CO2 cells were transferred to low CO2 conditions, indicating that ATP produced by cyclic electron transfer was necessary to accumulate DIC in low-CO2 conditions.

Cloning and characterization of a Chlamydomonas reinhardtii cDNA arylalkylamine N-acetyltransferase and its use in the genetic engineering of melatonin content in the Micro-Tom tomato.

Abstract:  Melatonin is found in a wide variety of plant species. Several investigators have studied the physiological roles of melatonin in plants. However, its role is not well understood because of the limited information on its biosynthetic pathway. To clarify melatonin biosynthesis in plants, we isolated a cDNA‐coded arylalkylamine N‐acetyltransferase (AANAT), a possible limiting enzyme for melatonin biosynthesis, from Chlamydomonas reinhardtii (designated as CrAANAT). The predicted amino acid sequence of CrAANAT shares 39.0% homology to AANAT from Ostreococcus tauri and lacks cAMP‐dependent protein kinase phosphorylation sites in the N‐ and C‐terminal regions that are conserved in vertebrates. The enzyme activity of CrAANAT was confirmed by in vitro assay using Escherichia coli. Transgenic plants constitutively expressing the CrAANAT were produced using Micro‐Tom, a model cultivar of tomato (Solanum lycopersicum L.). The transgenic Micro‐Tom exhibited higher melatonin content compared with wild type, suggesting that melatonin was synthesized from serotonin via N‐acetylserotonin in plants. Moreover, the melatonin‐rich transgenic Micro‐Tom can be used to elucidate the role of melatonin in plant development.

Physiological responses of lipids in Emiliania huxleyi and Gephyrocapsa oceanica (Haptophyceae) to growth status and their implications for alkenone paleothermometry

Abstract The physiological responses of alkenone unsaturation indices to changes in growth status of E. huxleyi and G. oceanica strains isolated from a water sample of the NW Pacific were examined using an isothermal batch culture system. In both E. huxleyi and G. oceanica the unsaturation index U37K′ changed during the growth period, but the effects of this change were different. This suggests that genotypic variation rather than the adaptation of the strains to the geographical environment of the sampling location is a major factor in determining the physiological responses to U37K′. Changes of U37K′ were associated with those of the unsaturation indices of C38 and C39 alkenones, the abundance ratios of lower to higher homologues of alkenones, the abundance ratios of saturated to polyunsaturated n-fatty acids, the abundance ratio of ethyl alkenoate to alkenones, and sterol contents. These associations might be attributable to the physiological response of lipids for maintaining their fluidity. The degree of unsaturation both in alkenones and n-fatty acids increased at day 8, possibly due to nutrient depletion. The ethyl alkenoate/total alkenone and ethyl alkenoate/C37 alkenone ratios increased abruptly at day 8 in both strains. These ratios should be useful in clarifying the relationship between the marine environment and its corresponding growth phase of batch culture. E. huxleyi and G. oceanica can be effectively distinguished using the U37K′-U38EtK diagram.

Wavelength specificity of growth, photosynthesis, and hydrocarbon production in the oil-producing green alga Botryococcus braunii

The effect of monochromatic light on growth, photosynthesis, and hydrocarbon production was tested in Botryococcus braunii Bot-144 (race B), which produces triterpenoid hydrocarbons. The growth was higher in order of red, blue, and green light. The color of red light-grown cells became more orange-yellow and their shape dominantly changed to grape-like with long branches. Photosynthetic carbon fixation activity was higher in order of blue, red, and green light-grown cells, but photosystem activities showed no difference. In the pulse-chase experiments with (14)CO(2), no major difference was observed in the production of lipids, hydrocarbons, polysaccharides, or proteins among the three kinds of cells, although hydrocarbon production was slightly lower in green light-grown cells. These results indicate that blue and red light were more effective for growth, photosynthetic CO(2) fixation, and hydrocarbon production than green light, and that red light is the most efficient light source when calculated based on photoenergy supplied.

Selenium utilization strategy by microalgae.

The diversity of selenoproteins raises the question of why so many life forms require selenium. Selenoproteins are found in bacteria, archaea, and many eukaryotes. In photosynthetic microorganisms, the essential requirement for selenium has been reported in 33 species belonging to six phyla, although its biochemical significance is still unclear. According to genome databases, 20 species are defined as selenoprotein-producing organisms, including five photosynthetic organisms. In a marine coccolithophorid, Emiliania huxleyi (Haptophyta), we recently found unique characteristics of selenium utilization and novel selenoproteins using 75Se-tracer experiments. In E. huxleyi, selenite, not selenate, is the main substrate used and its uptake is driven by an ATP-dependent high-affinity, active transport system. Selenite is immediately metabolized to low-molecular mass compounds and partly converted to at least six selenoproteins, named EhSEP1–6. The most (EhSEP2) and second-most abundant selenoproteins (EhSEP1) are disulfide isomerase (PDI) homologous protein and thioredoxin reductase (TR) 1, respectively. Involvement of selenium in PDI is unique in this organism, while TR1 is also found in other organisms. In this review, we summarize physiological, biochemical, and molecular aspects of selenium utilization by microalgae and discuss their strategy of selenium utilization.

Cyanobacterial Lactate Oxidases Serve as Essential Partners in N2 Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants

This article analyses the evolution of a major photorespiratory enzyme, glycolate oxidase (GOX), finding that plant GOX was phylogenetically derived from cyanobacterial lactate oxidase. Cyanobacterial GOX-like proteins are today found only in N2-fixing strains, in which they play an important role in the protection of nitrogenase. Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N2-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high l-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N2-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N2 fixation was more sensitive to O2 in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O2-scavenging enzyme to protect nitrogenase in extant N2-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration.

Proteomic Analysis of High-CO2-Inducible Extracellular Proteins in the Unicellular Green Alga, Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii can acclimate to a wide range of CO(2) concentrations through the regulation of a CO(2)-concentrating mechanism (CCM). By proteomic analysis, here we identified the proteins which were specifically accumulated under high-CO(2) conditions in a cell wall-less strain of C. reinhardtii which release their extracellular matrix into the medium. When the CO(2) concentration was elevated from the ambient air level to 3% during culture, the algal growth rate increased 1.5-fold and the composition of extracellular proteins, but not intracellular soluble and insoluble proteins, clearly changed. Proteomic analysis data showed that the levels of 22 of 129 extracellular proteins increased for 1 and 3 d and such multiple high-CO(2)-inducible proteins include gametogenesis-related proteins and hydroxyproline-rich glycoproteins. However, we could not prove the induction of gametogenesis under high-CO(2) conditions, suggesting that the inductive signal might be incomplete, not strong enough or that only high-CO(2) conditions might be not sufficient for the cell stage to proceed to the formation of sexually active gametes. However, these gametogenesis-related proteins and/or hydroxyproline-rich glycoproteins may have novel roles outside the cell under high-CO(2) conditions.

Biofuels as a sustainable energy source: An update of the applications of proteomics in bioenergy crops and algae ☆

Sustainable energy is the need of the 21st century, not because of the numerous environmental and political reasons but because it is necessary to human civilizations energy future. Sustainable energy is loosely grouped into renewable energy, energy conservation, and sustainable transport disciplines. In this review, we deal with the renewable energy aspect focusing on the biomass from bioenergy crops to microalgae to produce biofuels to the utilization of high-throughput omics technologies, in particular proteomics in advancing our understanding and increasing biofuel production. We look at biofuel production by plant- and algal-based sources, and the role proteomics has played therein. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Optimization of light for growth, photosynthesis, and hydrocarbon production by the colonial microalga Botryococcus braunii BOT-22.

Optimization of the light conditions for biofuel production by the microalga Botryococcus braunii BOT-22 (race B) was performed using monochromatic red light. The lipid and sugar contents were approximately 40% and 20-30% of the cell dry weight, respectively, and about half of the lipids were liquid hydrocarbons. The half-saturation intensities for the production rate of lipids, hydrocarbons, and sugars were 63, 49, and 44μmolm(-2)s(-1), respectively. Fluorescence microscopic images of Nile Red-stained cells showed an increased number of intracellular neutral lipid granules due to increased light intensity. After 16days of incubation in the dark, lipid and sugar, but not hydrocarbon content decreased. Growth, metabolite production, and photosynthesis were saturated at 100, 200 and 1000μmolm(-2)s(-1), respectively. These results indicate that photosynthetically captured energy is not used efficiently for metabolite production; thus, improvements in metabolic regulation may increase hydrocarbon production.