Dongyuan Zhang

Chromosomal localization of 5S and 18S-5.8S-25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization.

Abstract.Fluorescence in situ hybridization (FISH) was employed on mitotic metaphase chromosome preparations of five Asian Pinus species: Pinus tabuliformis, Pinus yunnanensis, Pinus densata, Pinus massoniana and Pinus merkusii, using simultaneously DNA probes of the 18S rRNA gene and the 5S rRNA gene including the non-transcribed spacer sequences. The number and location of 18S rDNA sites varied markedly (5–10 pairs of strong signals) among the five pines. A maximum of 20 major 18S rDNA sites was observed in the diploid genome (2n = 24) of P. massoniana. The 5S rDNA FISH pattern was less variable, with one major site and one minor site commonly observed in each species. The differentiation of rDNA sites on chromosomes among the five pines correlates well with their phylogenic positions in Pinus as reconstructed from other molecular data. P. densata, a species of hybrid origin, resembles its parents (P. tabuliformis and P. yunnanensis), including some components characteristic of each parent in its pattern. However, the species is unique, showing new features resulting possibly from recombination and genome reorganization.

Genome-Wide Identification, Evolutionary Expansion, and Expression Profile of Homeodomain-Leucine Zipper Gene Family in Poplar (Populus trichocarpa)

Background Homeodomain-leucine zipper (HD-ZIP) proteins are plant-specific transcriptional factors known to play crucial roles in plant development. Although sequence phylogeny analysis of Populus HD-ZIPs was carried out in a previous study, no systematic analysis incorporating genome organization, gene structure, and expression compendium has been conducted in model tree species Populus thus far. Principal Findings In this study, a comprehensive analysis of Populus HD-ZIP gene family was performed. Sixty-three full-length HD-ZIP genes were found in Populus genome. These Populus HD-ZIP genes were phylogenetically clustered into four distinct subfamilies (HD-ZIP I–IV) and predominately distributed across 17 linkage groups (LG). Fifty genes from 25 Populus paralogous pairs were located in the duplicated blocks of Populus genome and then preferentially retained during the sequential evolutionary courses. Genomic organization analyses indicated that purifying selection has played a pivotal role in the retention and maintenance of Populus HD-ZIP gene family. Microarray analysis has shown that 21 Populus paralogous pairs have been differentially expressed across different tissues and under various stresses, with five paralogous pairs showing nearly identical expression patterns, 13 paralogous pairs being partially redundant and three paralogous pairs diversifying significantly. Quantitative real-time RT-PCR (qRT-PCR) analysis performed on 16 selected Populus HD-ZIP genes in different tissues and under both drought and salinity stresses confirms their tissue-specific and stress-inducible expression patterns. Conclusions Genomic organizations indicated that segmental duplications contributed significantly to the expansion of Populus HD-ZIP gene family. Exon/intron organization and conserved motif composition of Populus HD-ZIPs are highly conservative in the same subfamily, suggesting the members in the same subfamilies may also have conservative functionalities. Microarray and qRT-PCR analyses showed that 89% (56 out of 63) of Populus HD-ZIPs were duplicate genes that might have been retained by substantial subfunctionalization. Taken together, these observations may lay the foundation for future functional analysis of Populus HD-ZIP genes to unravel their biological roles.

Comprehensive analysis of CCCH zinc finger family in poplar (Populus trichocarpa)

BackgroundCCCH zinc finger proteins contain a typical motif of three cysteines and one histidine residues and serve regulatory functions at all stages of mRNA metabolism. In plants, CCCH type zinc finger proteins comprise a large gene family represented by 68 members in Arabidopsis and 67 in rice. These CCCH proteins have been shown to play diverse roles in plant developmental processes and environmental responses. However, this family has not been studied in the model tree species Populus to date.ResultsIn the present study, a comprehensive analysis of the genes encoding CCCH zinc finger family in Populus was performed. Using a thorough annotation approach, a total of 91 full-length CCCH genes were identified in Populus, of which most contained more than one CCCH motif and a type of non-conventional C-X11-C-X6-C-X3-H motif was unique for Populus. All of the Populus CCCH genes were phylogeneticly clustered into 13 distinct subfamilies. In each subfamily, the gene structure and motif composition were relatively conserved. Chromosomal localization of these genes revealed that most of the CCCHs (81 of 90, 90 %) are physically distributed on the duplicated blocks. Thirty-four paralogous pairs were identified in Populus, of which 22 pairs (64.7 %) might be created by the whole genome segment duplication, whereas 4 pairs seem to be resulted from tandem duplications. In 91 CCCH proteins, we also identified 63 putative nucleon-cytoplasm shuttling proteins and 3 typical RNA-binding proteins. The expression profiles of all Populus CCCH genes have been digitally analyzed in six tissues across different developmental stages, and under various drought stress conditions. A variety of expression patterns of CCCH genes were observed during Populus development, of which 34 genes highly express in root and 22 genes show the highest level of transcript abundance in differentiating xylem. Quantitative real-time RT-PCR (RT-qPCR) was further performed to confirm the tissue-specific expression and responses to drought stress treatment of 12 selected Populus CCCH genes.ConclusionsThis study provides the first systematic analysis of the Populus CCCH proteins. Comprehensive genomic analyses suggested that segmental duplications contribute significantly to the expansion of Populus CCCH gene family. Transcriptome profiling provides first insights into the functional divergences among members of Populus CCCH gene family. Particularly, some CCCH genes may be involved in wood development while others in drought tolerance regulation. Our results presented here may provide a starting point for the functional dissection of this family of potential RNA-binding proteins.

Increased lipid productivity and TAG content in Nannochloropsis by heavy-ion irradiation mutagenesis

One mutant (HP-1) with higher growth rate was obtained from Nannochloropsis oceanica IMET1 by heavy-ion irradiation mutagenesis. Compared to the wild type, the biomass accumulation and maximum growth rate of HP-1 were individually increased by 19% and 6%, and its lipid productivity was increased by 28% from 211 to 271 mg L(-1) d(-1). Subsequently analysis indicated photosynthetic efficiency of HP-1 was higher than that of wild type during cultivation. Further, lipid composition analysis indicated TAG content of HP-1 was 14% higher, while polar lipid content was 15% lower than that of wild type. Moreover, fatty acid profiles analysis revealed no significant variation was found between the two strains. The mutant is discussed in terms of its comparative advantage over the wild type with respect to its potential utilization for biodiesel production. Owing to its higher lipid productivity and TAG content, HP-1 could be considered as a valuable candidate for microalgal biodiesel production.

Purification and characterization of a new β-glucosidase from Penicillium piceum and its application in enzymatic degradation of delignified corn stover

A new β-glucosidase (Cel3B) was first isolated from cellulytic fungi, designated as PpCel3B. Although PpCel3B was classified to GH family 3 based on the homology sequence, PpCel3B had different biological functions in cellulose degradation and signaling molecules production. PpCel3B was constitutive and could form multiple soluble lignocellulose inducers for cellulase and hemicellulase synthesis via high tranglycosylation activity and new enzymatic activity. Moreover, PpCel3B showed apparent synergism with cellulases by removing several inhibitors. Supplementing low doses of PpCel3B (52 μg/g substrate) increased saccharification efficiency of cellulase produced by Trichoderma reesei and Penicillium piceum by 15% and 35%, respectively on delignified corn stover. PpCel3B had important application in boosting cellulase yield and efficiency.

HCF243 encodes a chloroplast-localized protein involved in the D1 protein stability of the arabidopsis photosystem II complex.

Numerous auxiliary nuclear factors have been identified to be involved in the dynamics of the photosystem II (PSII) complex. In this study, we characterized the high chlorophyll fluorescence243 (hcf243) mutant of Arabidopsis (Arabidopsis thaliana), which shows higher chlorophyll fluorescence and is severely deficient in the accumulation of PSII supercomplexes compared with the wild type. The amount of core subunits was greatly decreased, while the outer antenna subunits and other subunits were hardly affected in hcf243. In vivo protein-labeling experiments indicated that the synthesis rate of both D1 and D2 proteins decreased severely in hcf243, whereas no change was found in the rate of other plastid-encoded proteins. Furthermore, the degradation rate of the PSII core subunit D1 protein is higher in hcf243 than in the wild type, and the assembly of PSII is retarded significantly in the hcf243 mutant. HCF243, a nuclear gene, encodes a chloroplast protein that interacts with the D1 protein. HCF243 homologs were identified in angiosperms with one or two copies but were not found in lower plants and prokaryotes. These results suggest that HCF243, which arose after the origin of the higher plants, may act as a cofactor to maintain the stability of D1 protein and to promote the subsequent assembly of the PSII complex.

Enhanced hydrolysis of Macrocystis pyrifera by integrated hydroxyl radicals and hot water pretreatment.

Integrated hydroxyl radicals and hot water pretreatment (IHRHW) was employed in the bioconversion of the brown macroalgae Macrocystis pyrifera (M. pyrifera) in this study. The optimum experimental pretreatment condition (100°C, 30 min, 11.9 mM FeSO4) and the predicted optimum pretreatment condition (113.95°C, 29.1 min, 12.75 mM FeSO4) were identified using a central composite design method. All glucan and xylan were recovered as monosaccharides or polysaccharides without a fermentation inhibitor (e.g., hydroxymethyl furfural and furfural). The IHRHW-treated macroalgae digestibility reached 88.1% under the optimum experimental condition, whereas that under the predicted optimum condition reached 92.1%. The value was approximately threefold higher than those obtained with untreated M. pyrifera. Carbohydrate recovery and enzymatic hydrolysis can be significantly enhanced by the new economic hydroxyl radicals and hot water pretreatment.

FtsHi4 Is Essential for Embryogenesis Due to Its Influence on Chloroplast Development in Arabidopsis

Chloroplast formation is associated with embryo development and seedling growth. However, the relationship between chloroplast differentiation and embryo development remains unclear. Five FtsHi genes that encode proteins with high similarity to FtsH proteins, but lack Zn2+-binding motifs, are present in the Arabidopsis genome. In this study, we showed that T-DNA insertion mutations in the Arabidopsis FtsHi4 gene resulted in embryo arrest at the globular-to-heart–shaped transition stage. Transmission electron microscopic analyses revealed abnormal plastid differentiation with a severe defect in thylakoid formation in the mutant embryos. Immunocytological studies demonstrated that FtsHi4 localized in chloroplasts as a thylakoid membrane-associated protein, supporting its essential role in thylakoid membrane formation. We further showed that FtsHi4 forms protein complexes, and that there was a significant reduction in the accumulation of D2 and PsbO (two photosystem II proteins) in mutant ovules. The role of FtsHi4 in chloroplast development was confirmed using an RNA-interfering approach. Additionally, mutations in other FtsHi genes including FtsHi1, FtsHi2, and FtsHi5 caused phenotypic abnormalities similar to ftshi4 with respect to plastid differentiation during embryogenesis. Taken together, our data suggest that FtsHi4, together with FtsHi1, FtsHi2, and FtsHi5 are essential for chloroplast development in Arabidopsis.

Genome sequence of Talaromyces piceus 9-3 provides insights into lignocellulose degradation

Many species of Penicillium have exhibited great potential for lignocellulose hydrolysis. The filamentous fungus Talaromyces piceus 9-3 (anamorph: Penicillium piceum), which was isolated from compost wastes in China, was sequenced in this study. Compared with the cellulase producer T. reesei, T. piceus 9-3 processes a lignocellulolytic enzyme system comprising more diverse enzymatic components, especially hemicellulases. This report will facilitate the use of this strain for biomass degradation.

A β-glucosidase Hyperproducing Strain, Pencillium piceum: Novel Characterization of Lignocellulolytic Enzyme Systems and Its Application in Biomass Bioconversion

In this chapter, the β-glucosidase over producing strain Pencillium piceum and the β-glucosidase characteristics will be introduced. Through several rounds of dimethyl sulfate mutagenesis, the β-glucosidase activity of P. piceum reached 53.12 IU/ml. Two new β-glucosidases, promising bifunctional enzymes for lignocellulosic bioconversion, have been found in the extracellular protein of P. piceum. The two new β-glucosidases played an important role in forming multiple soluble cellulose inducers via high transglycosylation activity and novel enzymatic activity. Further, the two new β-glucosidases showed the strong synergism with different cellulases by removing multiple inhibitors for cellulase. The rational computer-aided strategies were devised to enhance the thermostability of the main β-glucosidase (Cel3A) from Penicillium piceum H16. Pencillium piceum, high-yielding β-glucosidase with high enzymatic activity and good thermostability, may provide a good synergetic effect on Trichoderma reesei for improving cellulose hydrolysis of different substrates.