Global transcriptome analysis and characterization of Dryopteris fragrans (L.) Schott sporangium in different developmental stages.

BACKGROUND: Dryopteris fragrans (D. fragrans) is a potential medicinal fern distributed in volcanic magmatic rock areas under tough environmental condition. Sporangia are important organs for fern reproduction. This study was designed to characterize the transcriptome characteristics of the wild D. fragrans sporangia in three stages (stage A, B, and C) with the aim of uncovering its molecular mechanism of growth and development. RESULTS: Using a HiSeq 4000, 79.81 Gb clean data (each sample is at least 7.95 GB) were obtained from nine samples, with three being supplied from each period, and assembled into 94,705 Unigenes, among which 44,006 Unigenes were annotated against public protein databases (NR, Swiss-Prot, KEGG, COG, KOG, GO, eggNOG and Pfam). Furthermore, we observed 7126 differentially expressed genes (DEG) (Fold Change > 4, FDR < 0.001), 349,885 SNP loci, and 10,584 SSRs. DEGs involved in DNA replication and homologous recombination were strongly expressed in stage A, and several DEGs involved in cutin, suberin and wax biosynthesis had undergone dramatic changes during development, which was consistent with morphological observations. DEGs responsible for secondary metabolism and plant hormone signal transduction changed clearly in the last two stages. DEGs homologous to those known genes associated with the development of reproductive organs of flowering plants have also been validated and discussed, such as AGL61, AGL62, ONAC010. In particular, a Unigene encoding TFL1, an important flower-development regulator in flowering plants, was identified and exhibited the highest expression level in stage B in D. fragrans sporangia. CONCLUSIONS: This study is the first report on global transcriptome analysis in the development of sporangia of wild D. fragrans. DEGs related to development and homologous to flower-seed development in flowering plants were discussed. All DEGs involved in DNA replication and homologous recombination were consistent with morphological observations of paraffin slices. The results of this study provide rare resources for further investigation of the D. fragrans sporangium development, stress resistance and secondary metabolism.

Halomonas songnenensis sp. nov., a moderately halophilic bacterium isolated from saline and alkaline soils.

A moderately halophilic bacterium (strain NEAU-ST10-39T) was isolated from saline and alkaline soils in the oilfield of Daqing City, Heilongjiang Province, China. The strain was strictly aerobic, Gram-stain-negative, rod-shaped and motile by peritrichous flagella. Its colonies were yellow. It grew at NaCl concentrations of 0.2-15% (w/v) (optimum 4%, w/v), at temperatures of 4-40 °C (optimum 35 °C) and at pH 5-10 (optimum pH 7). It did not produce acids from sugars or alcohols. Its DNA G+C content was 57.4 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated 16S rRNA, gyrB and rpoD gene sequences indicated that it belonged to the genus Halomonas in the class Gammaproteobacteria. The most phylogenetically related species were Halomonas axialensis, Halomonas meridiana and Halomonas aquamarina, whose types shared 98.3% (16S rRNA), 82.7% (gyrB) and 83.9-84.5% (rpoD) sequence similarity with strain NEAU-ST10-39T. The results of DNA-DNA hybridization assays showed 20±2%-50±1 % relatedness between strain NEAU-ST10-39T and the most closely related species including Halomonas axialensis DSM 15723T, Halomonas meridiana DSM 5425T, Halomonas aquamarina DSM 30161(T), Halomonas johnsoniae DSM 21197T, Halomonas stevensii DSM 21198T, Halomonas nanhaiensis CCTCC AB 2012911(T), Halomonas hamiltonii DSM 21196T and Halomonas arcis CGMCC 1.6494T. The major fatty acids were C18 : 1ω7c (47.2%), C16:1ω7c and/or C16:1ω6c (18.9%) and C16:0 (16.3%), the only respiratory quinone detected was ubiquinone 9 and polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown phospholipids and three unknown lipids. The new isolate is proposed to represent a novel species with the name Halomonas songnenensis sp. nov., NEAU-ST10-39T (=CGMCC 1.12152T=DSM 25870T) being the type strain.

Halomonas zhaodongensis sp. nov., a slightly halophilic bacterium isolated from saline-alkaline soils in Zhaodong, China.

A slightly halophilic bacterium (strain NEAU-ST10-25(T)) was isolated from saline-alkaline soils in Zhaodong City, Heilongjiang Province, China. The strain is a Gram-negative, aerobic motile rod. It accumulates poly-ß-hydroxyalkanoate and produces exopolysaccharide. It produces beige-yellow colonies. Growth occurs at NaCl concentrations (w/v) of 0-15 % (optimum 3 %), at temperatures of 4-60 °C (optimum 35 °C) and at pH 6-12 (optimum pH 9). Its G+C content is 53.8 mol%. Phylogenetic analyses based on the separate 16S rRNA gene and concatenation of the 16S rRNA, gyrB and rpoD genes indicate that it belongs to the genus Halomonas in the class Gammaproteobacteria. The most phylogenetically related species is Halomonas alkaliphila DSM 16354(T), with which strain NEAU-ST10-25(T) showed 16S rRNA, gyrB and rpoD gene sequence similarities of 99.2, 82.3 and 88.2 %, respectively. The results of DNA-DNA hybridization assays showed 60.47 ± 0.69 % DNA relatedness between strain NEAU-ST10-25(T) and H. alkaliphila DSM 16354(T), 42.43 ± 0.37 % between strain NEAU-ST10-25(T) and Halomonas venusta DSM 4743(T) and 30.62 ± 0.43 % between strain NEAU-ST10-25(T) and Halomonas hydrothermalis DSM 15725(T). The major fatty acids are C18:1 ω7c (62.3 %), C16:0 (17.6 %), C16:1 ω7c/C16:1 ω6c (7.7 %), C14:0 (2.9 %), C12:0 3-OH (2.8 %), C10:0 (2.1 %) and C18:1 ω9c (1.6 %) and the predominant respiratory quinone is ubiquinone 9 (Q-9). The proposed name is Halomonas zhaodongensis, NEAU-ST10-25(T) (=CGMCC 1.12286(T) = DSM 25869(T)) being the type strain.

TaEXPB7-B, a ß-expansin gene involved in low-temperature stress and abscisic acid responses, promotes growth and cold resistance in Arabidopsis thaliana.

Low temperature is one of the primary causes of economic loss in agricultural production, and in this regard, expansin proteins are known to play important roles in plant growth and responses to various abiotic stresses and plant hormones. In order to elucidate the roles of expansin genes in the response of Dongnongdongmai 2 (D2), a highly cold-resistant winter wheat variety, to low-temperature stress, we exposed plants to a temperature of 4℃ and analysed the transcriptome of tillering nodes. Expression levels of TaEXPB7-B were significantly increased in response to both low-temperature stress and abscisic acid (ABA) treatment. To further confirm these observations, we transformed Arabidopsis plants with the ß-glucuronidase (GUS) gene driven by the TaEXPB7-B promoter. GUS staining results revealed that TaEXPB7-B showed similar responses to low-temperature and ABA treatments. Our transcriptome data indicated that the AREB/ABF transcription factor gene TaWABI5 was also induced by low temperature in D2. Yeast one-hybrid experiments demonstrated that TaWABI5 binds to an ABRE cis-element in the TaEXPB7-B promoter, and overexpression of TaWABI5 in wheat protoplasts enhanced the expression of endogenous TaEXPB7-B by 7.7-fold, implying that TaWABI5 plays important roles in regulating the expression of TaEXPB7-B. Cytological data obtained from the transient expression of 35S::TaEXPB7-B-eYFP in onion epidermal cells indicated that TaEXPB7-B is cell wall localised. Overexpression of TaEXPB7-B in Arabidopsis promoted a significant increase in plant growth and increased lignin and cellulose contents. Moreover, TaEXPB7-B conferred enhanced antioxidant and osmotic regulation in transgenic Arabidopsis, thereby increasing the tolerance and survival of plants under conditions of low-temperature stress.

Screening of potential IL-tolerant cellulases and their efficient saccharification of IL-pretreated lignocelluloses.

Lignocellulosic biomass as one of the most abundant and renewable resources has great potential for biofuel production. The complete conversion of biomass to biofuel is achieved through the effective pretreatment process and the following enzyme saccharification. Ionic liquids (ILs) are considered as a green solvent for lignocellulose pretreatment. However, ILs exhibit an inhibitory effect on cellulase activity, leading to a subsequent decrease in the efficiency of saccharification. The screening of new potential IL-tolerant cellulases is important. In the current study, a fungal strain with a relatively high cellulase production was isolated and identified as Penicillium oxalicum HC6. The culture conditions were optimized using corn stover and peptone as the carbon source and nitrogen source at pH 4.0 and 30 °C with an inoculation size of 2% (v/v) for 8 days. It was found that P. oxalicum HC6 exhibited potential salt tolerance with the increase of the enzyme production at a salt concentration of 5.0% (w/v). In addition, high enzyme activities were obtained at pH 4.0-6.0 and 50-65 °C. The crude enzyme from P. oxalicum HC6 with good thermal stability was also stable in the presence of salt and ILs. Good yields of reducing sugar were obtained by the crude enzyme from P. oxalicum HC6 after the saccharification of corn stover that was pretreated by ILs. P. oxalicum HC6 with potentially salt-tolerant and IL-tolerant enzymes has great potential application in the enzymatic saccharification of lignocellulose.

Genome-wide identification of wheat (Triticum aestivum) expansins and expansin expression analysis in cold-tolerant and cold-sensitive wheat cultivars.

Plant expansins are proteins involved in cell wall loosening, plant growth, and development, as well as in response to plant diseases and other stresses. In this study, we identified 128 expansin coding sequences from the wheat (Triticum aestivum) genome. These sequences belong to 45 homoeologous copies of TaEXPs, including 26 TaEXPAs, 15 TaEXPBs and four TaEXLAs. No TaEXLB was identified. Gene expression and sub-expression profiles revealed that most of the TaEXPs were expressed either only in root tissues or in multiple organs. Real-time qPCR analysis showed that many TaEXPs were differentially expressed in four different tissues of the two wheat cultivars-the cold-sensitive 'Chinese Spring (CS)' and the cold-tolerant 'Dongnongdongmai 1 (D1)' cultivars. Our results suggest that the differential expression of TaEXPs could be related to low-temperature tolerance or sensitivity of different wheat cultivars. Our study expands our knowledge on wheat expansins and sheds new light on the functions of expansins in plant development and stress response.

Correction: De novo assembly and analysis of the transcriptome of Rumex patientia L. during cold stress.

[This corrects the article DOI: 10.1371/journal.pone.0186470.].

De novo assembly and analysis of the transcriptome of Rumex patientia L. during cold stress.

BACKGROUND: Rumex patientia L. is consumed as a green vegetable in several parts of the world, and can withstand extremely low temperatures (-35°C). However, little or no available genomic data for this species has been reported to date. Here, we used Illumina Hiseq technology for transcriptome assembly in R. patientia under normal and cold conditions to evaluate how it responds to cold stress. RESULTS: After an in-depth RNA-Seq analysis, 115,589 unigenes were produced from the assembled transcripts. Based on similarity search analysis with seven databases, we obtained and annotated 60,157 assembled unigenes to at least one database. In total, 1,179 unigenes that were identified as differentially expressed genes (DEGs), including up-regulated (925) and down-regulated ones (254), were successfully assigned GO annotations and classified into three major metabolic pathways. Ribosome, carbon metabolism, oxidative phosphorylation and biosynthesis of amino acids were the most highly enriched pathways according to KEGG analysis. Overall, 66 up-regulated genes were identified as putatively involved in the response to cold stress, including members of MYB, AP2/ERF, CBF, Znf, bZIP, NAC and COR families. CONCLUSION: To our knowledge, this investigation was the first to provide a cold-responsive (COR) transcriptome assembly in R. patientia. A large number of potential COR genes were identified, suggesting that this species is suitable for cultivation in northern China. In summary, these data provide valuable information for future research and genomic studies in R. patientia.

Genome-wide characterization of GRAS family genes in Medicago truncatula reveals their evolutionary dynamics and functional diversification.

The GRAS gene family is a large plant-specific family of transcription factors that are involved in diverse processes during plant development. Medicago truncatula is an ideal model plant for genetic research in legumes, and specifically for studying nodulation, which is crucial for nitrogen fixation. In this study, 59 MtGRAS genes were identified and classified into eight distinct subgroups based on phylogenetic relationships. Motifs located in the C-termini were conserved across the subgroups, while motifs in the N-termini were subfamily specific. Gene duplication was the main evolutionary force for MtGRAS expansion, especially proliferation of the LISCL subgroup. Seventeen duplicated genes showed strong effects of purifying selection and diverse expression patterns, highlighting their functional importance and diversification after duplication. Thirty MtGRAS genes, including NSP1 and NSP2, were preferentially expressed in nodules, indicating possible roles in the process of nodulation. A transcriptome study, combined with gene expression analysis under different stress conditions, suggested potential functions of MtGRAS genes in various biological pathways and stress responses. Taken together, these comprehensive analyses provide basic information for understanding the potential functions of GRAS genes, and will facilitate further discovery of MtGRAS gene functions.

Application of alkyl polyglycoside surfactant in ultrasonic-assisted extraction followed by macroporous resin enrichment for the separation of vitexin-2″-O-rhamnoside and vitexin from Crataegus pinnatifida leaves.

An alkyl polyglycoside (APG) surfactant was used in ultrasonic-assisted extraction to effectively extract vitexin-2″-O-rhamnoside (VOR) and vitexin (VIT) from Crataegus pinnatifida leaves. APG0810 was selected as the surfactant. The extraction process was optimized for ultrasonic power, the APG concentration, ultrasonic time, soaking time, and liquid-solid ratio. The proposed approach showed good recovery (99.80-102.50% for VOR and 98.83-103.19% for VIT) and reproducibility (relative standard deviation, n=5; 3.7% for VOR and 4.2% for VIT) for both components. The proposed sample preparation method is both simple and effective. The use of APG for extraction of key herbal ingredients shows great potential. Ten widely used commercial macroporous resins were evaluated in a screening study to identify a suitable resin for the separation and purification of VOR and VIT. After comparing static and dynamic adsorption and desorption processes, HPD100B was selected as the most suitable resin. After column adsorption and desorption on this resin, the target compounds VOR and VIT can be effectively separated from the APG0810 extraction solution. Recoveries of VOR and VIT were 89.27%±0.42% and 85.29%±0.36%, respectively. The purity of VOR increased from 35.0% to 58.3% and the purity of VIT increased from 12.5% to 19.9%.

[Identification of RAPD markers linked to the resistance gene Yr5 against wheat stripe rust with denaturing PAGE-silver staining].

RAPD analysis was performed between a near-isogenic line (NIL) Yr5/6 x Avocet S carrying the resistance gene Yr5 against wheat stripe rust and its susceptible parent Avocet S, using the Yr5 gene donor parent Triticum spelta album as control. Amplified DNA fragments were separated on 4% denaturing PAGE (polyacrylamide gel electrophoresis) and displayed by silver staining. Fifty to 100 bands were detected, 5 folds more than those revealed on agarose gels. A total of 240 random primers were screened, and 23 reproducible polymorphic DNA fragments were found, out of which 6 polymorphic bands appeared to be linked to Yr5 gene. Genetic linkage was tested on 121 segregating F2 plants derived from a cross between Avocet S and Yr5/6 x Avocet S. It was showed that the polymorphic DNA fragment S1320(207) was completely linked to Yr5 gene, and S1348(363) closely linked to Yr5 gene. The results suggested that using denaturing PAGE-silver staining could increase the level of DNA polymorphisms detected in wheat and also improve the repeatability of RAPD analysis.

Functional ionic liquids for hydrolysis of lignocellulose.

An efficient system for hydrolysis of lignocellulosic materials to prepare reducing sugar in a series of functional acidic ionic liquids with low synthetic cost and excellent dissolved and catalytic activity was established. High yield of reducing sugar was obtained with the use of 1-H-3-methylimidazolium chloride ([HMIM]Cl). The use of ionic liquid under ultrasound irradiation greatly improved the yield of total reducing sugar. The optimum reaction conditions were as follows: ratio of water/sample was 5 (w/w), ratio of IL/sample was 25 (w/w), 70°C, 120 min and the yield of reducing sugar was up to 53.27 mg from 0.2g of soybean straw and 50.03 mg from 0.2g of corn straw.