The pentatricopeptide repeat protein EMPTY PERICARP8 is required for the splicing of three mitochondrial introns and seed development in maize.

Splicing of plant organellar group II introns is under accurate nuclear control that employs many nucleus-encoded protein cofactors from various families. For mitochondrial introns, only a few splicing factors have been characterized because disruption of their functions often causes embryo lethality. Here, we report the function of Empty Pericarp8 (Emp8) in the splicing of three group II introns in mitochondria, complex I biogenesis, and seed development in maize. Emp8 encodes a P subfamily pentatricopeptide repeat protein that localizes in mitochondria. The loss-of-function mutants of Emp8 are embryo lethal, showing severely arrested embryo and endosperm development in maize. The respiration rate in the emp8 mutants is reduced with substantially enhanced expression of alternative oxidases. Transcript analysis indicated that the trans-splicing of nad1 intron 4 and cis-splicing of nad4 intron 1 are abolished, and the cis-splicing of nad2 intron 1 is severely impaired in the emp8 mutants. These defects consequently lead to the disassembly of mitochondrial complex I and a dramatic reduction in its activity. Together, these results suggest that Emp8 is required for the trans-splicing of nad1 intron 4 and cis-splicing of nad4 intron 1 and nad2 intron 1, which is essential to mitochondrial complex I assembly and hence to embryogenesis and endosperm development in maize.

Empty pericarp7 encodes a mitochondrial E-subgroup pentatricopeptide repeat protein that is required for ccmFN editing, mitochondrial function and seed development in maize.

RNA editing, converting cytidines (C) to uridines (U) at specific sites in the transcripts of mitochondria and plastids, plays a critical role in organelle gene expression in land plants. Recently pentatricopeptide repeat (PPR) proteins were identified as site-specific recognition factors for RNA editing. In this study, we characterized an empty pericarp7 mutant (emp7) in Zea mays (maize), which confers an embryo-lethal phenotype. In emp7 mutants, mitochondrial functions are seriously perturbed, resulting in a strikingly reduced respiration rate. Emp7 encodes an E-subgroup PPR protein that is localized exclusively in the mitochondrion. Null mutation of Emp7 abolishes the C → U editing of ccmF(N) transcript solely at position 1553. CcmF(N) is coding for a subunit of heme lyase complex in the cytochrome c maturation pathway. The resulting Phe → Ser substitution in CcmF(N) leads to the loss of CcmF(N) protein and a strikingly reduced c-type cytochrome. Consequently, the mitochondrial cytochrome-linked respiratory chain is impaired as a result of the disassembly of complex III in the emp7 mutant. These results indicate that the PPR-E subgroup protein EMP7 is required for C → U editing of ccmF(N) -1553 at a position essential for cytochrome c maturation and mitochondrial oxidative phosphorylation, and hence is essential to embryo and endosperm development in maize.

Comparative Transcriptome Analysis of Vibrio splendidus JZ6 Reveals the Mechanism of Its Pathogenicity at Low Temperatures.

Yesso scallop-pathogenic Vibrio splendidus strain JZ6 was found to have the highest virulence at 10°C, while its pathogenicity was significantly reduced with increased temperature and completely incapacitated at 28°C. In the present study, comparative transcriptome analyses of JZ6 and another nonpathogenic V. splendidus strain, TZ19, were conducted at two crucial culture temperatures (10°C and 28°C) in order to determine the possible mechanism of temperature regulation of virulence. Comparisons among four libraries, constructed from JZ6 and TZ19 cultured at 10°C and 28°C (designated JZ6_10, JZ6_28, TZ19_10, and TZ19_28), revealed that 241 genes were possibly related to the increased virulence of JZ6 at 10°C. There were 10 genes, including 2 encoding Flp pilus assembly proteins (FlhG and VS_2437), 6 encoding proteins of the "Vibrio cholerae pathogenic cycle" (ToxS, CqsA, CqsS, RpoS, HapR, and Vsm), and 2 encoding proteins in the Sec-dependent pathway (SecE and FtsY), that were significantly upregulated in JZ6_10 (P < 0.05) compared to those in JZ6_28, TZ19_10, and TZ19_28, which were supposed to be responsible for adhesion, quorum sensing, virulence, and protein secretion of V. splendidus. When cultured at 10°C, JZ6 cells were larger and tended to aggregate more than those cultured at 28°C. The virulence factor (extracellular metalloprotease) was also found to be highly expressed in the extracellular product (ECP) of JZ6 at 10°C, and this ECP exhibited obvious cytotoxicity to oyster primary hemocytes, A549 cells, and L929 cells. These results indicated that low temperatures (10°C) could enhance adhesion, activate the quorum sensing systems, upregulate virulence factor synthesis and secretion, and, lastly, increase the pathogenicity of JZ6.

Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis.

Substrate binding is typically one of the rate-limiting steps preceding enzyme catalytic action during homogeneous reactions. However, interfacial-based enzyme catalysis on insoluble crystalline substrates, like cellulose, has additional bottlenecks of individual biopolymer chain decrystallization from the substrate interface followed by its processive depolymerization to soluble sugars. This additional decrystallization step has ramifications on the role of enzyme-substrate binding and its relationship to overall catalytic efficiency. We found that altering the crystalline structure of cellulose from its native allomorph I(ß) to III(I) results in 40-50% lower binding partition coefficient for fungal cellulases, but surprisingly, it enhanced hydrolytic activity on the latter allomorph. We developed a comprehensive kinetic model for processive cellulases acting on insoluble substrates to explain this anomalous finding. Our model predicts that a reduction in the effective binding affinity to the substrate coupled with an increase in the decrystallization procession rate of individual cellulose chains from the substrate surface into the enzyme active site can reproduce our anomalous experimental findings.

The comprehensive immunomodulation of NeurimmiRs in haemocytes of oyster Crassostrea gigas after acetylcholine and norepinephrine stimulation.

BACKGROUND: Neural-endocrine-immune (NEI) system is a major modulation network among the nervous, endocrine and immune system and weights greatly in maintaining homeostasis of organisms during stress and infection. Some microRNAs are found interacting with NEI system (designated NeurimmiRs), addressing swift modulations on immune system. The oyster Crassostrea gigas, as an intertidal bivalve, has evolved a primary NEI system. However, the knowledge about NeurimmiRs in oysters remains largely unknown. RESULTS: Six small RNA libraries from haemocytes of oysters stimulated with acetylcholine (ACh) and norepinephrine (NE) were sequenced to identify neurotransmitter-responsive miRNAs and survey their immunomodulation roles. A total of 331 miRNAs (132 identified in the present study plus 199 identified previously) were subjected to expression analysis, and twenty-one and sixteen of them were found ACh- or NE-responsive, respectively (FDR < 0.05). Meanwhile, 21 miRNAs exhibited different expression pattern after ACh or NE stimulation. Consequently, 355 genes were predicted as putative targets of these neurotransmitter-responsive miRNAs in oyster. Through gene onthology analysis, multiple genes involved in death, immune system process and response to stimulus were annotated to be modulated by NeurimmiRs. Besides, a significant decrease in haemocyte phagocytosis and late-apoptosis or necrosis rate was observed after ACh and NE stimulation (p < 0.05) while early-apoptosis rate remained unchanged. CONCLUSIONS: A comprehensive immune-related network involving PRRs, intracellular receptors, signaling transducers and immune effectors was proposed to be modulated by ACh- and NE-responsive NeurimmiRs, which would be indispensable for oyster haemocytes to respond against stress and infection. Characterization of the NeurimmiRs would be an essential step to understand the NEI system of invertebrate and the adaptation mechanism of oyster.

Preparation of Epoxy Resin with Disulfide-Containing Curing Agent and Its Application in Self-Healing Coating.

Intrinsic self-healing polymers via dynamic covalent bonds have been attracting extensive attention because of their repeatable self-healing property. Herein, a novel self-healing epoxy resin was synthesized with disulfide-containing curing agent via the condensation of dimethyl 3,3'-dithiodipropionate (DTPA) and polyether amine (PEA). Therefore, in the structure of cured resin, flexible molecular chains and disulfide bonds were imported into the cross-linked polymer networks for triggering self-healing performance. The self-healing reaction of cracked samples was realized under a mild condition (60 °C for 6 h). The distribution of flexible polymer segments, disulfide bonds and hydrogen bonds in cross-linked networks plays a great role in the self-healing process of prepared resins. The molar ratio of PEA and DTPA strongly affects the mechanical performance and self-healing property. Especially when that molar ratio of PEA to DTPA is 2, the cured self-healing resin sample showed great ultimate elongation (795%) and excellent healing efficiency (98%). The products can be used as an organic coating, in which the crack could self-repair during a limited time. The corrosion resistance of a typical cure coating sample has been testified by an immersion experiment and electrochemistry impedance spectrum (EIS). This work provided a simple and low-cost route to prepare a self-healing coating for prolonging the service life of conventional epoxy coatings.

Identification of Respiratory Burst Oxidase Homolog (Rboh) Family Genes From Pyropia yezoensis and Their Correlation With Archeospore Release.

Reactive oxygen species (ROS) play important regulatory roles in plant growth and development, as well as in cell differentiation and stress responses. Respiratory burst oxidase homolog (RBOH) is the key enzyme in ROS production. So far, the Rboh family genes in Pyropia yezoensis have not been comprehensively characterized, and whether their function was involved in the formation of archeospores is still unknown. In this study, a total of 11 PyRboh genes were identified from the P. yezoensis genome by homology mining. Through phylogenetic analysis, it is suggested that the PyRboh genes were evolutionarily conserved among the lineages of red algae, but a few genes exhibited a species-specific manner. The treatment of P. yezoensis blades with NADPH oxidase inhibitor diphenylene iodonium (DPI) could significantly inhibit the formation of archeospores, suggesting that RBOH may be involved in the formation of archeospores. According to PyRboh gene expression analysis using the P. yezoensis strains with obvious differences in releasing archeospores, it is showed that the expression trends of most genes were consistent, with no significant difference between strains, whereas the expression pattern of the two P. yezoensis-specific genes (PyRbohJ and PyRbohK) was positively correlated with the amount of archeospores. Furthermore, as treatment of blades with allantoin resulted in a significant increase in the release of archeospores, the expression levels of PyRbohJ and PyRbohK were also consistently upregulated, further confirming the relationship between the two genes and archeospore formation. These findings provide insights into the molecular mechanism of P. yezoensis archeospore formation.

Proteomics-based compositional analysis of complex cellulase-hemicellulase mixtures.

Efficient deconstruction of cellulosic biomass to fermentable sugars for fuel and chemical production is accomplished by a complex mixture of cellulases, hemicellulases, and accessory enzymes (e.g., >50 extracellular proteins). Cellulolytic enzyme mixtures, produced industrially mostly using fungi like Trichoderma reesei, are poorly characterized in terms of their protein composition and its correlation to hydrolytic activity on cellulosic biomass. The secretomes of commercial glycosyl hydrolase-producing microbes was explored using a proteomics approach with high-throughput quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we show that proteomics-based spectral counting approach is a reasonably accurate and rapid analytical technique that can be used to determine protein composition of complex glycosyl hydrolase mixtures that also correlates with the specific activity of individual enzymes present within the mixture. For example, a strong linear correlation was seen between Avicelase activity and total cellobiohydrolase content. Reliable, quantitative and cheaper analytical methods that provide insight into the cellulosic biomass degrading fungal and bacterial secretomes would lead to further improvements toward commercialization of plant biomass-derived fuels and chemicals.

Restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate.

Conversion of lignocellulose to biofuels is partly inefficient due to the deleterious impact of cellulose crystallinity on enzymatic saccharification. We demonstrate how the synergistic activity of cellulases was enhanced by altering the hydrogen bond network within crystalline cellulose fibrils. We provide a molecular-scale explanation of these phenomena through molecular dynamics (MD) simulations and enzymatic assays. Ammonia transformed the naturally occurring crystalline allomorph I(ß) to III(I), which led to a decrease in the number of cellulose intrasheet hydrogen bonds and an increase in the number of intersheet hydrogen bonds. This rearrangement of the hydrogen bond network within cellulose III(I), which increased the number of solvent-exposed glucan chain hydrogen bonds with water by ~50%, was accompanied by enhanced saccharification rates by up to 5-fold (closest to amorphous cellulose) and 60-70% lower maximum surface-bound cellulase capacity. The enhancement in apparent cellulase activity was attributed to the "amorphous-like" nature of the cellulose III(I) fibril surface that facilitated easier glucan chain extraction. Unrestricted substrate accessibility to active-site clefts of certain endocellulase families further accelerated deconstruction of cellulose III(I). Structural and dynamical features of cellulose III(I), revealed by MD simulations, gave additional insights into the role of cellulose crystal structure on fibril surface hydration that influences interfacial enzyme binding. Subtle alterations within the cellulose hydrogen bond network provide an attractive way to enhance its deconstruction and offer unique insight into the nature of cellulose recalcitrance. This approach can lead to unconventional pathways for development of novel pretreatments and engineered cellulases for cost-effective biofuels production.

Binding characteristics of Trichoderma reesei cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass.

Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput fast protein liquid chromatography (HT-FPLC)-based method has been developed to quantify cellobiohydrolase I (CBH I or Cel7A), cellobiohydrolase II (CBH II or Cel6A), and endoglucanase I (EG I or Cel7B) present in hydrolyzates of untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated corn stover (CS). This method can accurately quantify individual enzymes present in complex binary and ternary protein mixtures without interference from plant cell wall-derived components. The binding isotherms for CBH I, CBH II, and EG I were obtained after incubation for 2 h at 4 °C. Both AFEX and dilute acid pretreatment resulted in increased cellulase binding compared with untreated CS. Cooperative binding of CBH I and/or CBH II in the presence of EG I was observed only for AFEX treated CS. Competitive binding between enzymes was found for certain other enzyme-substrate combinations over the protein loading range tested (i.e., 25-450 mg/g glucan). Langmuir single-site adsorption model was fitted to the binding isotherm data to estimate total available binding sites E(bm) (mg/g glucan) and association constant K(a) (L/mg). Our results clearly demonstrate that the characteristics of cellulase binding depend not only on the enzyme GH family but also on the type of pretreatment method employed.

Identification of SFR6, a key component in cold acclimation acting post-translationally on CBF function.

The sfr6-1 mutant of Arabidopsis thaliana was identified previously on the basis of its failure to undergo acclimation to freezing temperatures following exposure to low positive temperatures. This failure is attributed to a defect in the pathway leading to cold on-regulated (COR) gene expression via CBF (C-box binding factor) transcription factors. We identified a region of chromosome 4 containing SFR6 by positional mapping. Fine mapping of the sfr6-1 mutation proved impossible as the locus resides very close to the centromere. Therefore, we screened 380 T-DNA lines with insertions in genes within the large region to which sfr6-1 mapped. This resulted in the identification of two further mutant alleles of SFR6 (sfr6-2 and sfr6-3); like the original sfr6-1 mutation, these disrupt freezing tolerance and COR gene expression. To determine the protein sequence, we cloned an SFR6 cDNA based on the predicted coding sequence, but this offered no indication as to the mechanism by which SFR6 acts. The SFR6 gene itself is not strongly regulated by cold, thus discounting regulation of SFR6 activity at the transcriptional level. We show that over-expression of CBF1 or CBF2 transcription factors, which constitutively activate COR genes in the wild-type, cannot do so in sfr6-1. We demonstrate that CBF protein accumulates to wild-type levels in response to cold in sfr6-1. These results indicate a role for the SFR6 protein in the CBF pathway -downstream of CBF translation. The fact that the SFR6 protein is targeted to the nucleus may suggest a direct role in modulating gene expression.

Inhibition of glutathione synthesis decreases chilling tolerance in Chorispora bungeana callus.

The possible roles of reduced glutathione (GSH) in chilling tolerance were studied in callus generated from a representative alpine plant, Chorispora bungeana Fisch. & C.A. Mey (C. bungeana). The callus grew well under low-temperature and chilling treatment led only to slight injury, as indicated by a low level of ion leakage (IL). Malondialdehyde measurements also were not elevated, however GSH was. Exogenously application of l-buthionine-(S R)-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase (gamma-ECS), arrested the GSH accumulation induced by chilling and resulted in a significant decrease in cell growth and an increase in IL and malondialdehyde. These results implied that C. bungeana is a plant with a strong low-temperature tolerance mechanism, and the tolerance of C. bungeana may be associated with GSH accumulation. Under chilling treatment, the proportion of unsaturated fatty acid in the plasma membrane (PM) increased significantly in callus of C. bungeana mainly due to increases in C18:2 and C18:3, the membrane fluidity (indicated by DPH fluorescent polarization) however was maintained. High PM H(+)-ATPase activities were also induced by chilling. Exogenously application of BSO blocked the effects of chilling treatments on the changes of fatty acids and PM H(+)-ATPase activities, reducing the PM membrane fluidity. On the other hand, simultaneous application of GSH and BSO to callus under chilling treatments reversed the effects of BSO on the changes of fatty acids, PM fluidity and PM H(+)-ATPase activities. These results suggested that GSH induced by low-temperature treatments may confer chilling tolerance to C. bungeana, probably by increasing unsaturated fatty acid compositions and maintaining PM fluidity and high enzymatic activity.

Formation of capillary tube-like structures on micropatterned biomaterials.

The survival of three-dimensional tissue requires a vascular network to provide transport of oxygen and metabolic byproduct. Here, we report a new approach to create capillary blood vessels in vitro on biomaterials suitable for use as scaffolds in engineering tissues. Endothelial cells were cultured on chemical and topographical patterns of micro-sized grooves on gelatin. Selective attachment and spreading of cells within the grooves was ensured by microcontact printing the plateau regions with cell resistant PEG/PLA (polyethyleneglycol-L-polylacticacid). Human microvascular endothelial cells plated on these patterned biomaterials attached and spread exclusively within the grooves. These topographical features promote endothelial cells to form capillary tube-like structures. The results demonstrated that capillary structures formed on biomaterials are useful for engineering vascularized tissues.

Characterization of the complete chloroplast genome of Caulerpa cupressoides (Bryopsidales, Chlorophyta).

Caulerpa cupressoides (Vahl) C. Agardh is a widely distributed tropical green algae. The circular chloroplast genome was 130,895 bp in length, with a GC content of 34%. In total, 99 genes were identified and they were consisted of 63 coding genes, 30 tRNA genes, and 6 rRNA genes. This chloroplast genome did not show an obvious quadripartite structure. Phylogenetic analysis revealed that C. cupressoides, C. racemosa, and Tydemania expeditionis were close relatives, with high bootstrap values. The characterized complete chloroplast genome of C. cupressoides will provide essential date for further studies of Bryopsidales.

Characterization of the whole chloroplast genome Caulerpa lentillifera J. Agardh (Bryopsidales, Chlorophyta).

The whole chloroplast genome (cp DNA) sequence of Caulerpa lentillifera J. Agardh has been characterized from Illumina pair-end sequencing. The circular cpDNA was 119,402 bp in length, containing 122 genes, which included 91 protein-coding genes, 28 tRNA genes, and 3 ribosomal RNA genes (four rRNA species). The overall AT content of C. lentillifera cpDNA is 67.4%. The 48 genes phylogenetic analysis suggested that C. lentillifera formed a monophyletic clade with congeneric C. racemosa.

Extraction, characterization and biological activity of sulfated polysaccharides from seaweed Dictyopteris divaricata.

Dictyopteris divaricata is a kind of important brown algae with many biological activities. It has been receiving more and more attention, yet there are rarely studies done on its polysaccharides. In this study, the optimum extraction and biological activity of seaweed polysaccharides from Dictyopteris divaricata (DDSP) were investigated. Response surface methodology (RSM), based on a three-level, three-variable Box-Behnken design (BBD), was employed to obtain the best possible combinations for maximum polysaccharides yield. The optimum extraction conditions were as follows: liquid-solid ratio of 110 mL/g, extraction time of 6 h and extraction temperature of 100 °C. Under these conditions, the experimental yield was 3.05%, which was in close agreement with the predicted value of 3.15%. The average molecular weight of DDSP was 58.05 kDa. Gas chromatograph (GC) results showed that DDSP was composed of fucose, xylose, mannose, glucose and galactose with the corresponding molar ratio of 4.45:2.74:1.00:2.94:1.35. Biological activity showed that DDSP exhibited strong antioxidant activity in vitro and possessed the potential on stimulating immune response of RAW264.7 cells. So DDSP can be used as a natural ingredient in functional foods.

Adaptive Transcriptome Profiling of Subterranean Zokor, Myospalax baileyi, to High- Altitude Stresses in Tibet.

Animals living at high altitudes have evolved distinct phenotypic and genotypic adaptations against stressful environments. We studied the adaptive patterns of altitudinal stresses on transcriptome turnover in subterranean plateau zokors (Myospalax baileyi) in the high-altitude Qinghai-Tibetan Plateau. Transcriptomes of zokors from three populations with distinct altitudes and ecologies (Low: 2846 m, Middle: 3282 m, High: 3,714 m) were sequenced and compared. Phylogenetic and principal component analyses classified them into three divergent altitudinal population clusters. Genetic polymorphisms showed that the population at H, approaching the uppermost species boundary, harbors the highest genetic polymorphism. Moreover, 1056 highly up-regulated UniGenes were identified from M to H. Gene ontologies reveal genes like EPAS1 and COX1 were overexpressed under hypoxia conditions. EPAS1, EGLN1, and COX1 were convergent in high-altitude adaptation against stresses in other species. The fixation indices (F ST and G ST )-based outlier analysis identified 191 and 211 genes, highly differentiated among L, M, and H. We observed adaptive transcriptome changes in Myospalax baileyi, across a few hundred meters, near the uppermost species boundary, regardless of their relatively stable underground burrows' microclimate. The highly variant genes identified in Myospalax were involved in hypoxia tolerance, hypercapnia tolerance, ATP-pathway energetics, and temperature changes.

Draft Genome Sequence of Alcanivorax sp. Strain KX64203 Isolated from Deep-Sea Sediments of Iheya North, Okinawa Trough.

This report describes the draft genome sequence of Alcanivorax sp. strain KX64203, isolated from deep-sea sediment samples. The reads generated by an Ion Torrent PGM were assembled into contigs, with a total size of 4.76 Mb. The data will improve our understanding of the strain's function in alkane degradation.

Draft Genome Sequences of Pseudoalteromonas telluritireducens DSM 16098 and P. spiralis DSM 16099 Isolated from the Hydrothermal Vents of the Juan de Fuca Ridge.

This report describes the draft genome sequences of two strains, Pseudoalteromonas telluritireducens DSM 16098 and P. spiralis DSM 16099, which were isolated from hydrothermal vents of the Juan de Fuca Ridge. The reads generated by an Ion Torrent PGM were assembled into contigs with total sizes of 4.4 Mb and 4.1 Mb for DSM 16098 and DSM 16099, respectively.

The simple neuroendocrine-immune regulatory network in oyster Crassostrea gigas mediates complex functions.

The neuroendocrine-immune (NEI) regulatory network is a complex system, which plays an indispensable role in the immunity of the host. In the present study, the bioinformatical analysis of the transcriptomic data from oyster Crassostrea gigas and further biological validation revealed that oyster TNF (CgTNF-1 CGI_10018786) could activate the transcription factors NF-κB and HSF (heat shock transcription factor) through MAPK signaling pathway, and then regulate apoptosis, redox reaction, neuro-regulation and protein folding in oyster haemocytes. The activated immune cells then released neurotransmitters including acetylcholine, norepinephrine and [Met(5)]-enkephalin to regulate the immune response by arising the expression of three TNF (CGI_10005109, CGI_10005110 and CGI_10006440) and translocating two NF-κB (Cgp65, CGI_10018142 and CgRel, CGI_10021567) between the cytoplasm and nuclei of haemocytes. Neurotransmitters exhibited the immunomodulation effects by influencing apoptosis and phagocytosis of oyster haemocytes. Acetylcholine and norepinephrine could down-regulate the immune response, while [Met(5)]-enkephalin up-regulate the immune response. These results suggested that the simple neuroendocrine-immune regulatory network in oyster might be activated by oyster TNF and then regulate the immune response by virtue of neurotransmitters, cytokines and transcription factors.