Functional division of f-type and m-type thioredoxins to regulate the Calvin cycle and cyclic electron transport around photosystem I

Redox regulation of chloroplast proteins is necessary to adjust photosynthetic performance with changes in light. The thioredoxin (Trx) system plays a central role in this process. Chloroplast-localized classical Trx is a small redox-active protein that regulates many target proteins by reducing their disulfide bonds in a light-dependent manner. Arabidopsis thaliana mutants lacking f-type Trx (trx f1f2) or m-type Trx (trx m124-2) have been reported to show delayed reduction of Calvin cycle enzymes. As a result, the trx m124-2 mutant exhibits growth defects. Here, we characterized a quintuple mutant lacking both Trx f and Trx m to investigate the functional complementarity of Trx f and Trx m. The trx f1f2 m124-2 quintuple mutant was newly obtained by crossing, and is analyzed here for the first time. The growth defects of the trx m124-2 mutant were not enhanced by the lack of Trx f. In contrast, deficiencies of both Trxs additively suppressed the reduction of Calvin cycle enzymes, resulting in a further delay in the initiation of photosynthesis. Trx f appeared to be necessary for the rapid activation of the Calvin cycle during the early induction of photosynthesis. To perform effective photosynthesis, plants seem to use both Trxs in a coordinated manner to activate carbon fixation reactions. In contrast, the PROTON GRADIENT REGULATION 5 (PGR5)-dependent cyclic electron transport around photosystem I was regulated by Trx m, but not by Trx f. Lack of Trx f did not affect the activity and regulation of the PGR5-dependent pathway. Trx f may have a higher specificity for target proteins, whereas Trx m has a variety of target proteins to regulate overall photosynthesis and other metabolic reactions in the chloroplasts.

     

Protection of Coral Larvae from Thermally Induced Oxidative Stress by Redox Nanoparticles

Coral reefs are one of the most biologically diverse and economically important ecosystems on earth. However, the destruction of coral reefs has been reported worldwide owing to rising seawater temperature associated with global warming. In this study, we investigated the potential of a redox nanoparticle (RNP^O) to scavenge reactive oxygen species (ROS), which are overproduced under heat stress and play a crucial role in causing coral mortality. When reef-building coral (Acropora tenuis) larvae, without algal symbionts, were exposed to thermal stress at 33 °C, RNP^O treatment significantly increased the survival rate. Proteome analysis of coral larvae was performed using nano-liquid chromatography-tandem mass spectrometry for the first time. The results revealed that several proteins related to ROS-induced oxidative stress were specifically identified in A. tenuis larvae without RNP^O treatment, whereas these proteins were absent in RNP^O-treated larvae, which suggested that RNP^O effectively scavenged ROS from A. tenuis larvae. Results from this study indicate that RNP^O treatment can reduce ROS in aposymbiotic coral larvae and would be a promising approach for protecting corals from thermal stress.     

Carbohydrate-recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family

Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9.     

Cloning of cDNAs encoding cell-wall hydrolases from pear (Pyrus communis) fruit and their involvement in fruit softening and development of melting texture

'La France' pear ( Pyrus communis L.) fruit stored at 1°C for 1 month (short-term storage) before transfer to 20°C softened and developed a melting texture during ripening, whereas fruit stored for 5 months (long-term storage) before transfer to 20°C softened but did not develop a melting texture. To clarify the mechanisms involved in fruit softening and textural changes, the cDNAs encoding cell-wall hydrolases were isolated by RT-PCR, and their expression and localization were investigated in 'La France' pears. Genes encoding three polygalacturonases (PG; EC 3.2.1.15), four pectin methylesterases (PME; EC 3.1.1.11), one α -arabinofuranosidase (ARF; EC 3.2.1.55), three β -galactosidases (GAL; EC 3.2.1.23), and two endo-1,4- β - d -glucanases (Cel; EC 3.2.1.4) were isolated. Among these 13 isolated genes, PcPG1 was the only gene for which the mRNA expression levels increased in both the short- and long-term stored fruits. This suggested that PcPG1 is involved in fruit softening rather than in the development of the melting texture. In contrast, the expression levels of PcPG3 , PcPME1 , PcPME2 , PcPME3 , PcGAL1 , PcGAL2 , and PcCel2 increased during ripening only in the short-term stored fruit. These genes might thus be involved in the development of the melting texture.     

Phenotypic Plasticity in Photosynthetic Temperature Acclimation among Crop Species with Different Cold Tolerances

While interspecific variation in the temperature response of photosynthesis is well documented, the underlying physiological mechanisms remain unknown. Moreover, mechanisms related to species-dependent differences in photosynthetic temperature acclimation are unclear. We compared photosynthetic temperature acclimation in 11 crop species differing in their cold tolerance, which were grown at 15°C or 30°C. Cold-tolerant species exhibited a large decrease in optimum temperature for the photosynthetic rate at 360 μL L⁻¹ CO₂ concentration [Opt (A₃₆₀)] when growth temperature decreased from 30°C to 15°C, whereas cold-sensitive species were less plastic in Opt (A₃₆₀). Analysis using the C₃ photosynthesis model shows that the limiting step of A₃₆₀ at the optimum temperature differed between cold-tolerant and cold-sensitive species; ribulose 1,5-bisphosphate carboxylation rate was limiting in cold-tolerant species, while ribulose 1,5-bisphosphate regeneration rate was limiting in cold-sensitive species. Alterations in parameters related to photosynthetic temperature acclimation, including the limiting step of A₃₆₀, leaf nitrogen, and Rubisco contents, were more plastic to growth temperature in cold-tolerant species than in cold-sensitive species. These plastic alterations contributed to the noted growth temperature-dependent changes in Opt (A₃₆₀) in cold-tolerant species. Consequently, cold-tolerant species were able to maintain high A₃₆₀ at 15°C or 30°C, whereas cold-sensitive species were not. We conclude that differences in the plasticity of photosynthetic parameters with respect to growth temperature were responsible for the noted interspecific differences in photosynthetic temperature acclimation between cold-tolerant and cold-sensitive species.The temperature dependence of leaf photosynthetic rate shows considerable variation between plant species and with growth temperature (Berry and Björkman, 1980; Cunningham and Read, 2002; Hikosaka et al., 2006). Plants native to low-temperature environments and those grown at low temperatures generally exhibit higher photosynthetic rates at low temperatures and lower optimum temperatures, compared with plants native to high-temperature environments and those grown at high temperatures (Mooney and Billings, 1961; Slatyer, 1977; Berry and Björkman, 1980; Sage, 2002; Salvucci and Crafts-Brandner, 2004b). For example, the optimum temperature for photosynthesis differs between temperate evergreen species and tropical evergreen species (Hill et al., 1988; Read, 1990; Cunningham and Read, 2002). Such differences have been observed even among ecotypes of the same species (Björkman et al., 1975; Pearcy, 1977; Slatyer, 1977).Temperature dependence of the photosynthetic rate has been analyzed using the biochemical model proposed by Farquhar et al. (1980). This model assumes that the photosynthetic rate (A) is limited by either ribulose 1,5-bisphosphate (RuBP) carboxylation (A_c) or RuBP regeneration (A_r). The optimum temperature for photosynthetic rate in C₃ plants is thus potentially determined by (1) the temperature dependence of A_c, (2) the temperature dependence of A_r, or (3) both, at the colimitation point of A_c and A_r (Fig. 1; Farquhar and von Caemmerer, 1982; Hikosaka et al., 2006).Open in a separate windowFigure 1.A scheme illustrating the shift in the optimum temperature for photosynthesis depending on growth temperature. Based on the C₃ photosynthesis model, the A₃₆₀ (white and black circles) is limited by A_c (solid line) or A_r (broken line). The optimum temperature for the photosynthetic rate is potentially determined by temperature dependence of A_c (A), temperature dependence of A_r (B), or the intersection of the temperature dependences of A_c and A_r (C). When the optimum temperature for the photosynthetic rate shifts to a higher temperature, there are also three possibilities determining the optimum temperature: temperature dependence of A_c (D), temperature dependence of A_r (E), or the intersection of the temperature dependences of A_c and A_r (F). Especially in the case that the optimum temperature is determined by the intersection of the temperature dependences of A_c and A_r, the optimum temperature can shift by changes in the balance between A_c and A_r even when the optimum temperatures for these two partial reactions do not change.In many cases, the photosynthetic rate around the optimum temperature is limited by A_c, and thus the temperature dependence of A_c determines the optimum temperature for the photosynthetic rate (Hikosaka et al., 1999, 2006; Yamori et al., 2005, 2006a, 2006b, 2008; Sage and Kubien, 2007; Sage et al., 2008). As the temperature increases above the optimum, A_c is decreased by increases in photorespiration (Berry and Björkman, 1980; Jordan and Ogren, 1984; von Caemmerer, 2000). Furthermore, it has been suggested that the heat-induced deactivation of Rubisco is involved in the decrease in A_c at high temperature (Law and Crafts-Brandner, 1999; Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004a; Yamori et al., 2006b). Numerous previous studies have shown changes in the temperature dependence of A_c with growth temperature (Hikosaka et al., 1999; Bunce, 2000; Yamori et al., 2005). Also, the temperature sensitivity of Rubisco deactivation may differ between plant species (Salvucci and Crafts-Brandner, 2004b) and with growth temperature (Yamori et al., 2006b), which may explain variation in the optimum temperature for photosynthesis (Fig. 1, A and D).A_r is more responsive to temperature than A_c and often limits photosynthesis at low temperatures (Hikosaka et al., 1999, 2006; Sage and Kubien, 2007; Sage et al., 2008). Recently, several researchers indicated that A_r limits the photosynthetic rate at high temperature (Schrader et al., 2004; Wise et al., 2004; Cen and Sage, 2005; Makino and Sage, 2007). They suggested that the deactivation of Rubisco at high temperatures is not the cause of decreased A_c but a result of limitation by A_r. However, it remains unclear whether limitation by A_r is involved in the variation in the optimum temperature for the photosynthetic rate (Fig. 1, B and E).A shift in the optimum temperature for photosynthesis can result from changes in the balance between A_r and A_c, even when the optimum temperatures for these two partial reactions do not change (Fig. 1, C and F; Farquhar and von Caemmerer, 1982). The balance between A_r and A_c has been shown to change depending on growth temperature (Hikosaka et al., 1999; Hikosaka, 2005; Onoda et al., 2005a; Yamori et al., 2005) and often brings about a shift in the colimitation temperature of A_r and A_c. Furthermore, recent studies have shown that plasticity in this balance differs among species or ecotypes (Onoda et al., 2005b; Atkin et al., 2006; Ishikawa et al., 2007). Plasticity in this balance could explain interspecific variation in the plasticity of photosynthetic temperature dependence (Farquhar and von Caemmerer, 1982; Hikosaka et al., 2006), although there has been no evidence in the previous studies that the optimum temperature for photosynthesis occurs at the colimitation point of A_r and A_c.Temperature tolerance differs between species and, with growth temperature, even within species from the same functional group (Long and Woodward, 1989). Bunce (2000) indicated that the temperature dependences of A_r and A_c to growth temperature were different between species from cool and warm climates and that the balance between A_r and A_c was independent of growth temperature for a given plant species. However, it was not clarified what limited the photosynthetic rate or what parameters were important in temperature acclimation of photosynthesis. Recently, we reported that the extent of temperature homeostasis of leaf respiration and photosynthesis, which is assessed as a ratio of rates measured at their respective growth temperatures, differed depending on the extent of the cold tolerance of the species (Yamori et al., 2009b). Therefore, comparisons of several species with different cold tolerances would provide a new insight into interspecific variation of photosynthetic temperature acclimation and their underlying mechanisms. In this study, we selected 11 herbaceous crop species that differ in their cold tolerance (Yamori et al., 2009b) and grew them at two contrasting temperatures, conducting gas-exchange analyses based on the C₃ photosynthesis model (Farquhar et al., 1980). Based on these results, we addressed the following key questions. (1) Does the plasticity in photosynthetic temperature acclimation differ between cold-sensitive and cold-tolerant species? (2) Does the limiting step of photosynthesis at several leaf temperatures differ between plant species and with growth temperature? (3) What determines the optimum temperature for the photosynthetic rate among A_c, A_r, and the intersection of the temperature dependences of A_c and A_r?     

Molecular identification and characterization of an alginate-binding protein on the cell surface of Sphingomonas sp. A1

Cells of Sphingomonas sp. A1 (strain A1) directly incorporate a macromolecule, alginate, into cytoplasm through a biosystem, or "super-channel," consisting of a pit on the cell surface, alginate-binding proteins in periplasm, and an ABC transporter in the inner membrane. The pit functions as a concentrator for extracellular alginate. Through differential display analysis, a protein (p8) with a molecular mass of 20kDa and a pI of 7.4 was found to be inducibly expressed in the outer membrane of alginate-grown cells. The gene coding for p8 was identified in the genome of strain A1 and shown to be similar to that for the polyhydroxyalkanoate granule-associated protein of Ralstonia eutropha. The disruptant of p8 gene showed significant growth retardation in the alginate medium. An overexpression system for p8 was constructed in Escherichia coli, and the protein was purified and characterized. Surface plasmon resonance biosensor analysis indicated that p8 is able to bind alginate most efficiently at pH 4.0. The above results indicate that p8 is a cell surface protein able to bind alginate and facilitates the concentration of alginate in the pit on the cell surface of strain A1.     

Oxidative stress and delayed-onset muscle damage after exercise

Reactive oxygen species (ROS) produced during exercise may be involved in delayed-onset muscle damage related to inflammation. To investigate this hypothesis, we studied whether oxidative stress increases nuclear translocation of nuclear factor-kappaB and chemokine expression in skeletal muscle using myotube L6 cells. We also assessed whether prolonged acute exercise could increase these parameters in rats. In L6 cells, H(2)O(2) induced nuclear translocation of p65 and increased the expression of cytokine-induced neutrophil chemoattractant-1 (CINC-1) and monocyte chemoattractant protein-1 (MCP-1), whereas preincubation with alpha-tocopherol limited the increase in these proteins. Sprague Dawley rats were divided into the following groups: rested control, exercised, rested with a high alpha-tocopherol diet, and exercised with a high alpha-tocopherol diet. After 3 weeks of acclimation, both exercise groups ran on a treadmill at 25 m/min for 60 min. Exercise increased nuclear p65, CINC-1, and MCP-1 in gastrocnemius muscle cells, but these changes were ameliorated by the high alpha-tocopherol diet. Increases in myeloperoxidase and thiobarbituric acid-reactive substrates were ameliorated by a high alpha-tocopherol diet, as were the histological changes. Neutrophil activity was not altered by either exercise or a high alpha-tocopherol diet. These results indicate that delayed-onset muscle damage induced by prolonged exercise is partly related to inflammation via phagocyte infiltration caused by ROS and that alpha-tocopherol (an antioxidant) can attenuate such inflammatory changes.     

Environmental DNA enables detection of terrestrial mammals from forest pond water

Terrestrial animals must have frequent contact with water to survive, implying that environmental DNA (eDNA) originating from those animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. The versatility of MiMammal primers was tested in silico and by amplifying DNAs extracted from tissues. The results suggested that MiMammal primers are capable of amplifying and distinguishing a diverse group of mammalian species. In addition, analyses of water samples from zoo cages of mammals with known species composition suggested that MiMammal primers could successfully detect mammalian species from water samples in the field. Then, we performed an experiment to detect mammals from natural ecosystems by collecting five 500‐ml water samples from ponds in two cool‐temperate forests in Hokkaido, northern Japan. MiMammal amplicon libraries were constructed using eDNA extracted from water samples, and sequences generated by Illumina MiSeq were subjected to data processing and taxonomic assignment. We thereby detected multiple species of mammals common to the sampling areas, including deer (Cervus nippon), mouse (Mus musculus), vole (Myodes rufocanus), raccoon (Procyon lotor), rat (Rattus norvegicus) and shrew (Sorex unguiculatus). Many previous applications of the eDNA metabarcoding approach have been limited to aquatic/semiaquatic systems, but the results presented here show that the approach is also promising even for forest mammal biodiversity surveys.     

Involvement of MEK-ERK signaling pathway in the inhibition of cell growth by troglitazone in human pancreatic cancer cells

Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes, partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Asc1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation.