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As an important seedling source, monospores closely associate with yields in nori farming. However, the molecular mechanism underlying differences in monospore production for different strains remains unknown. Comparative transcriptome analysis was performed to examine gene expression differences between the spore abundant wild-type strain (WT) and spore deficient mutant (Y1) of Pyropia chauhanii. The WT strain that produces monospores in abundance exhibited more differentially expressed genes (DEGs) in both number and higher fold-changes than the Y1 strain incapable of producing monospores, indicating that the specific regulation of genes is involved in monospore production. Three lists of DEGs were obtained between the two strains using intersection and displayed in Venn diagram: one expressed only in WT strain, another expressed only in Y1 strain, and the third shared in both strains. DEGs annotated as homologous genes of Arabidopsis thaliana in these 3 lists were curated for online functional enrichment analysis on Metascape website. Gene regulatory networks of WT were functionally enriched in the processing, proteolysis, and transport of proteins, especially within the small GTPase protein family, which might be account for the monospore production ability, whereas Y1 were functionally enriched in the metabolism of essential substance and utilization of indispensable energy, which might be account for the rapid growth of blades. We found the differentially enriched gene regulatory networks between strains might be the intrinsic mechanisms of the different monospore production traits. These findings provide novel insights into the genes and regulatory networks associated with monospore production abilities, which are essential for developing accurate breeding technologies for optimal release of monospores and increase of total nori production.

Nori is the cultivated seaweed with the highest market value in the world. Monospores from the thalli reseed the cultivating nets and provide useful secondary seedlings in nori farming, but the underlying molecular basis remains unclear. To explore crucial genes and pathways related to monospore-producing process in Phycocalida chauhanii (Pyropia chauhanii), transcriptomic analysis over the time-course of key developmental stages was carried out. According to comparative analysis, a total of 19,490 differentially expressed genes (DEGs) were identified. Based on time series trend clustering analysis, 7294 DEGs were enriched in a predominant profile 45, in good accordance with the actual temporal monospore-producing processes, exhibiting an expression trend of first upregulated and then downregulated. Gene Ontology (GO) analysis revealed that the function of these DEGs were top three enriched in GO biological processes of positive regulation of GTPase activity, signal transduction, and protein phosphorylation. KEGG pathway analysis indicated that these DEGs were top three enriched in the “mitogen-activated protein kinase (MAPK) signaling pathway, regulation of autophagy, and endocytosis”. Besides, the expression levels of 12 selected DEGs were validated by real-time quantitative PCR and proved the accuracy of RNA-seq results. Collectively, these findings provide a comprehensive landscape of the molecular mechanism underlying the formation and release of monospores, which might serve as essential basis for the identification of key genes that contribute to monospore formation.

Calmodulin (CaM) plays an important regulatory role in plant growth and development. In this study, on the basis of previous transcriptomic annotation a homolog of CaM gene (Pc-CaM7) was identified from the red alga Phycocalida chauhanii (previously known as Porphyra chauhanii). It encoded a 151 amino acids protein with 4 EF-hand regions, which was the typical structure of calmodulin family proteins. Evolutionary analysis showed that Pc-CaM7 shared conserved orthologous relationships with CaMs from Neoporphyra haitanensis (formerly Pyropia haitanensis), Porphyra umbilicalis, and Gracilariopsis chorda. During the developmental period of P. chauhanii gametophytic germlings from 25- to 50-day-old, the asexual archeospores were continuedly released from the marginal region of blades, followed by the emergence of spermatangia from 40-day-old germlings. Based on quantitative real-time PCR analysis, it showed that expression trend of Pc-CaM7 was correlated with the rule of archeospores’ formation and release, whereas in N. haitanensis without archeospore characteristic, the expression pattern of the orthologous gene (Pha_00404) was different, which increased and reached a plateau during the same developmental period. These findings suggested that Pc-CaM7 may be involved in the process of archeospores formation and release, which may provide a foundation for understanding the molecular mechanisms of asexual reproduction in Bangiales.

As an economically important marine seaweed, Pyropia yezoensis can be asexually reproduced via archeospores. However, the molecular mechanism of archeospore formation remains unclear. In this study, we characterized the transcriptomes of two strains of Py. yezoensis, which are strain W′ with abundant archeospores and strain W with scarce archeospores. The 25- and 35-day-old gametophytic blades of both strains were sampled for RNA sequencing. Based on comparative transcriptomic analysis, a large number of differential expressed genes (DEGs) of four pairwise groups between samples were generated. In an effort to identify the genes related to archeospore formation, 415 consensus DEGs reflecting the difference between two strains were investigated. Gene ontology and KEGG analysis revealed that the function for these DEGs was enriched in various processes, such as translation, ribosome assembly, RNA methylation, vesicles assembly, intracellular localization of organelles, and endocytosis. According to functional analysis on 80 selected DEGs, it indicated that genes involved in calcium and MAPK signaling pathway, cytoskeletal movement, and cell wall degradation were related to the formation of archeospores. The accuracy of RNA-seq results was validated by quantitative real-time PCR of 8 selected DEGs. These findings suggest that the molecular mechanism of archeospore formation in Py. yezoensis is sophisticated and our results will serve as an essential foundation for further studies.

An edible green algal species Caulerpa lentillifera J. Agardh is reported from China for the first time. The species was collected from the southwest of Hainan Island and morphologically identified...

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.

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.

Bacterial disease has caused high mortality of breeding molluscs from 2009 to 2011 in the Changhai area (Dalian, China). Vibrio spp. and Pseudomonas spp. have been detected as major pathogenic agents for aquatic animals in this area. In the present study, four virulence genes including vsm, toxR, aprX and carA were targeted to develop a real-time PCR assay for the quantitative detection of Vibrio splendidus, V. parahaemolyticus, Pseudomonas fluorescens and P. putida, respectively. The sensitivity and specificity of the assays were verified by experimental samples, and the variation tendencies of pathogenic V. splendidus, V. parahaemolyticus, P. fluorescens and P. putida strains from June to September were also detected in mollusc farming waters during 2011-2014. The concentration of V. splendidus increased from June to July, reduced in August, and then increased again in September. The highest count of V. parahaemolyticus appeared in July, and then dramatically decreased from August to September. Conversely, the counts of P. fluorescens and P. putida remained at lower levels from June to August, and then dramatically peaked in September. All four pathogenic bacteria displayed similar fluctuation tendencies of count variation in each year, and their concentrations were found to have a correlation with the average annual temperature. The variation tendency of pathogenic bacteria with temperature suggested that temperature was one of the most important factors to regulate the bacterial growth in a farming area, which could further provide information for the early warning of disease outbreak by using a convenient real-time PCR assay.

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.

Tumor necrosis factor superfamily (TNFSF) members represent a group of cytokines participating in diverse immunological, pathological and developmental pathways. However, compared with deuterostomia and cnidaia, the composition and evolution of TNF homologous in protostomia are still not well understood. In the present study, a total of 81 TNF superfamily (TNFSF) genes from 15 mollusk species, including 23 TNFSF genes from Crassostrea gigas, were surveyed by genome-wide bioinformatics analysis. The phylogenetic analysis showed that 14 out of 23 C. gigas TNFSF genes in five clades exhibited orthologous relationships with Pinctada fucata TNFSF genes. Moreover, there were 15 C. gigas TNFSF genes located in oyster-specific clusters, which were contributed by small-scaled tandem and/or segmental duplication events in oyster. By comparing the sequences of duplicated TNFSF pairs, exon loss and variant in exon/intron length were revealed as the major modes of divergence in gene structure. Most of the duplicated C. gigas TNFSF pairs were evolved under purifying selection with consistent tissue expression patterns, implying functional constraint shaped diversification. This study demonstrated the expansion and early divergence of TNF superfamily in C. gigas, which provides potential insight into revealing the evolution and function of this superfamily in mollusk. (C) 2015 Elsevier Ltd. All rights reserved.

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.

Most cellulolytic enzyme blends, either procured from a commercial vendor or isolated from a single cellulolytic microbial secretome, do not efficiently hydrolyze ammonia-pretreated (e.g., ammonia fiber expansion or AFEX) lignocellulosic agricultural crop residues like corn stover to fermentable sugars. Typically reported commercial enzyme loading (30-100 mg protein/g glucan) necessary to achieve >90% total hydrolysis yield (to monosaccharides) for AFEX treated biomass, within a short saccharification time frame (24-48 h), is economically unviable. Unlike acid based pretreatments, AFEX retains most of the hemicelluloses in the biomass and therefore requires a more complex suite of enzymes for efficient hydrolysis of cellulose and hemicellulose at industrially relevant high solids loadings. One strategy to reduce enzyme dosage while improving cocktail effectiveness for AFEX treated biomass has been to use individually purified enzymes to determine optimal enzyme combinations to maximize hydrolysis yields. However, this approach is limited by the selection of heterologous enzymes available or the labor required for isolating low abundance enzymes directly from the microbial secretomes. Here, we show that directly blending crude cellulolytic and hemicellulolytic enzymes-rich microbial secretomes can maximize specific activity on AFEX treated biomass without having to isolate individual enzymes. Fourteen commercially available cellulolytic and hemicellulolytic enzymes were procured from leading enzyme companies (Novozymes®, Genencor®, and Biocatalysts®) and were mixed together to generate several hundred unique cocktail combinations. The mixtures were assayed for activity on AFEX treated corn stover (AFEX-CS) using a previously established high-throughput methodology. The optimal enzyme blend combinations identified from these screening assays were enriched in various low abundance hemicellulases and accessory enzymes typically absent in most commercial cellulases cocktails. Our simple approach of blending crude commercially available enzyme cocktails allowed a drastic four-fold reduction in total enzyme requirements (from 30 to 7.5 mg enzyme/g glucan loading) to achieve near-theoretical cellulose and hemicellulose saccharification yields for AFEX treated corn stover.

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.

Micro ribonucleic acids (miRNAs) represent a class of small noncoding RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or by repressing mRNA translation. In the case of algal lineages, especially dinoflagellates, knowledge regarding the miRNA system is still limited and its regulatory role remains unclear. In the present study, a computational approach was employed to screen miRNAs from the expressed sequence tags (ESTs) of Alexandrium tamarense. A total of 18 potential miRNAs were identified according to a range of filtering criteria. In addition, unique evolutionary features, such as miRNA gene duplication and sequence similarity to metazoan miRNAs, implied that the miRNA system in dinoflagellates is complex. Moreover, based on these 18 miRNA sequences, 42 potential target genes showing diverse functions in regulating growth and development were predicted in Thalassiosira pseudonana and Phaeodactylum tricornutum. Taken together, our data suggest the existence of miRNAs in dinoflagellates and provide clues for further functional studies on these predicted miRNAs.

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 β 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.

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.

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.

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 [Met5]-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 [Met5]-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.

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.

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.

We have applied a rational four-step strategy to identify novel bacterial glycosyl hydrolases (GH), in combination with various fungal enzymes, in order to define an efficient enzyme cocktail to hydrolyze pretreated lignocellulosic biomass. The fungal cellulases include cellobiohydrolase I (CBH I; GH family 7A), cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B) and ��-glucosidase (��G; GH family 3). Bacterial endocellulases (LC1 and LC2; GH family 5), ��-glucosidase (L��G; GH family 1), endoxylanases (LX1 and LX2; GH family 10) and ��-xylosidase (L��X; GH family 52) from multiple sources were cloned, expressed and purified as well. Enzymatic hydrolysis for various enzyme combinations was performed on Ammonia Fiber Expansion (AFEX) treated corn stover at various total protein loadings (30, 15 and 10 mg enzyme/g glucan). The optimal mass ratio of enzymes necessary for obtaining high glucan and xylan yields was determined using a suitable mixture design of experiments. The optimal hybrid enzyme mixtures contain fungal cellulases (78% of total protein loading), which include CBH I (loading ranging between 9-51% of total enzyme load), CBH II (9-51%), EG I (10-50%), and bacterial hemicellulases (22% of total protein loading) comprising of LXl (13%) and L��X (9%). The hybrid mixture works effectively at 50 ��C and pH 4.5 to efficiently saccharify AFEX treated corn stover giving as high as 95% glucan and 65% xylan conversion, respectively. This strategy of screening enzyme mixtures on pretreated lignocellulose will ultimately help develop enzyme cocktails that can hydrolyze plant cell walls efficiently and economically to produce cellulosic ethanol.

Pinus densata is a homoploid hybrid species, originating from P. tabuliformis × P. yunnanensis. The physiological fitness of this natural hybrid compared to its two parental species remains unknown. In this study, we investigated physiological responses of the three species by exposing artificially breed seedlings of each to drought stress lasting 28 days. Our results suggest that, in all three species, drought affected the contents of the plants’ chlorophyll, stomatal conductance, TBARS, hydrogen peroxide, and free proline and increased the activities of antioxidant enzymes, including superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), and peroxidase (POD; EC 1.11.1.7). The drought stress also induced significant changes in the activity of ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), dehydroascorbate reductase (DHAR; EC 1.8.5.1), glutathione reductase (GR; EC 1.6.4.2), and levels of ascorbate and glutathione in the ascorbate–glutathione cycle. The hybrid species P. densata appeared to achieve greater drought tolerance and exhibit hybrid superiority in antioxidant processes and other related physiological traits compared to the two parental species, although a few of the hybrid’s measured variables were similar to those of P. tabuliformis. However, P. yunnanensis was more sensitive to drought and appeared to have the lowest resistance to such stress. These physiological differences are largely consistent with the species’ habitat preferences, which may reflect their early genetic divergences and niche differentiation. These findings provide important information for management and forest restoration efforts of these species in the future.

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.

Gene duplication has been proposed to serve as the engine of evolutionary innovation. It is well recognized that eukaryotic genomes contain a large number of duplicated genes that evolve new functions or expression patterns. However, in mollusks, the evolutionary mechanisms underlying the divergence and the functional maintenance of duplicate genes remain little understood. In the present study, we performed a comprehensive analysis of duplicate genes in the protein kinase superfamily using whole genome and transcriptome data for the Pacific oyster. A total of 64 duplicated gene pairs were identified based on a phylogenetic approach and the reciprocal best BLAST method. By analyzing gene expression from RNA-seq data from 69 different developmental and stimuli-induced conditions (nine tissues, 38 developmental stages, eight dry treatments, seven heat treatments, and seven salty treatments), we found that expression patterns were significantly correlated for a number of duplicate gene pairs, suggesting the conservation of regulatory mechanisms following divergence. Our analysis also identified a subset of duplicate gene pairs with very high expression divergence, indicating that these gene pairs may have been subjected to transcriptional subfunctionalization or neofunctionalization after the initial duplication events. Further analysis revealed a significant correlation between expression and sequence divergence (as revealed by synonymous or nonsynonymous substitution rates) under certain conditions. Taken together, these results provide evidence for duplicate gene sequence and expression divergence in the Pacific oyster, accompanying its adaptation to harsh environments. Our results provide new insights into the evolution of duplicate genes and their expression levels in the Pacific oyster.

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.

The species-level taxonomy of the marine brown algal genus Sargassum is problematic. To resolve some of these systematic issues in the northern China seas, a molecular analysis of Sargassum species was carried out using ITS-2, cox3, and rbcL DNA sequences. Phylogenetic analyses confirmed eight species, including: S. confusum, S. fusiforme, S. hemiphyllum var. chinense, S. horneri, S. muticum, S. siliquastrum, S. thunbergii, and S. vachellianum. The previously described S. shandongense and S. qingdaoense, endemic to the Shandong Peninsula, are shown to be synonymous with S. vachellianum. Seven species previously described from the East China Sea were not observed, and require further investigation.

Thellungiella salsuginea (Brassiaceae) is a typical halophyte which can tolerate extreme cold, drought, and salinity. In order to understand the adaptive evolution of this species in the arid habitats, it is important to know its genetic structure. In this study, 17 polymorphic microsatellite loci were isolated and characterized from an enrichment genomic library of this species. We further assessed the polymorphisms of each locus in 18 individuals from nine geographically distant populations. The number of alleles per locus ranged from six to fourteen. The observed and expected heterozygosity ranged from 0.17 to 0.28 and 0.32 to 0.45, respectively. These markers have been crossly checked in another congeneric species, T. halophila. These microsatellite markers will be useful for investigating population genetics and adaptive evolution of this species and morphological divergence between and it and the closely related species.

In this work, six core glycosyl hydrolases (GH) were isolated and purified from various sources to help rationally optimize an enzyme cocktail to digest ammonia fiber expansion (AFEX) treated corn stover. The four core cellulases were endoglucanase I (EG I, GH family 7B), cellobiohydrolase I (CBH I, GH family 7A), cellobiohydrolase II (CBH II, GH family 6A) and ?-glucosidase (?G, GH family 3). The two core hemicellulases were an endo-xylanase (EX, GH family 11) and a ?-xylosidase (?X, GH family 3). Enzyme family and purity were confirmed by proteomics. Synergistic interactions among the six core enzymes for varying relative and total protein loading (8.25, 16.5 and 33 mg/g glucan) during hydrolysis of AFEX-treated corn stover was studied using a high-throughput microplate based protocol. The optimal composition (based on% protein mass loading) of the cocktail mixture was CBH I (28.4%): CBH II (18.0%): EG I (31.0%): EX (14.1%): ?G (4.7%): ?X (3.8%). These results demonstrate a rational strategy for the development of a minimal, synergistic enzymes cocktail that could reduce enzyme usage and maximize the fermentable sugar yields from pretreated lignocellulosics. � 2009 Elsevier Ltd.

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

Background Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification. Results In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansiona (AFEX™-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin. Conclusions Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.