Genome-Wide Analysis of WRKY Transcription Factors Involved in Abiotic Stress and ABA Response in Caragana korshinskii.

The WRKY transcription factor family plays a vital role in plant development and environmental response. However, the information of WRKY genes at the genome-wide level is rarely reported in Caragana korshinskii. In this study, we identified and renamed 86 CkWRKY genes, which were further classified into three groups through phylogenetic analysis. Most of these WRKY genes were clustered and distributed on eight chromosomes. Multiple sequence alignment revealed that the conserved domain (WRKYGQK) of the CkWRKYs was basically consistent, but there were also six variation types (WRKYGKK, GRKYGQK, WRMYGQK, WRKYGHK, WKKYEEK and RRKYGQK) that appeared. The motif composition of the CkWRKYs was quite conservative in each group. In general, the number of WRKY genes gradually increased from lower to higher plant species in the evolutionary analysis of 28 species, with some exceptions. Transcriptomics data and RT-qPCR analysis showed that the CkWRKYs in different groups were involved in abiotic stresses and ABA response. Our results provided a basis for the functional characterization of the CkWRKYs involved in stress resistance in C. korshinskii.

Multi-omics analysis reveals the molecular changes accompanying heavy-grazing-induced dwarfing of Stipa grandis.

Heavy grazing significantly reduces Stipa grandis growth. To enhance our understanding of plant responses to heavy grazing, we conducted transcriptomic, proteomic, and metabolic analyses of the leaves of non-grazed plants (NG) and heavy-grazing-induced dwarf plants (HG) of S. grandis. A total of 101 metabolites, 167 proteins, and 1,268 genes differed in abundance between the HG and NG groups. Analysis of Kyoto Encyclopedia of Genes and Genomes pathways among differentially accumulated metabolites (DAMs) revealed that the most enriched pathways were flavone and flavonol biosynthesis, tryptophan metabolism, and phenylpropanoid biosynthesis. An integrative analysis of differentially expressed genes (DEGs) and proteins, and DAMs in these three pathways was performed. Heavy-grazing-induced dwarfism decreased the accumulation of DAMs enriched in phenylpropanoid biosynthesis, among which four DAMs were associated with lignin biosynthesis. In contrast, all DAMs enriched in flavone and flavonol biosynthesis and tryptophan metabolism showed increased accumulation in HG compared with NG plants. Among the DAMs enriched in tryptophan metabolism, three were involved in tryptophan-dependent IAA biosynthesis. Some of the DEGs and proteins enriched in these pathways showed different expression trends. The results indicated that these pathways play important roles in the regulation of growth and grazing-associated stress adaptions of S. grandis. This study enriches the knowledge of the mechanism of heavy-grazing-induced growth inhibition of S. grandis and provides valuable information for restoration of the productivity in degraded grassland.

Selection and validation of reference genes for RT-qPCR analysis of different organs at various development stages in Caragana intermedia.

Reverse transcription quantitative PCR (RT-qPCR) is a technique widely used to investigate the expression of genes. An appropriate reference gene (RG) is essential for RT-qPCR analysis to obtain accurate and reliable results. Caragana intermedia plays an important role in afforestation as a bush. However, due to the lack of appropriate RGs, the research on development-related genes is limited. In this study, the selection for suitable RGs of different organs at various development stages to normalize the results of RT-qPCR about development-related genes was performed. To test the expression stability across all samples, we used the software algorithms such as geNorm, NormFinder, BestKeeper, and RefFinder to evaluate all the candidate RGs. Our results showed that CiEF1α was the most stable RG with little fluctuation among all samples. In addition, CiGAPDH in roots, CiSKIP1 in stems and leaves, and CiEF1α in different organs were selected as the most stable RGs. To confirm the applicability of the most stable RGs, the relative expression of CiWRKY17 was normalized using different candidate RGs. Taken together, our research laid a foundation for the study of development-related genes in C. intermedia.

Cloning and prokaryotic expression of WRKY48 from Caragana intermedia.

Caragana intermedia (C. intermedia) is a kind of drought-tolerant leguminous shrub. WRKY transcription factors are one of the largest family of transcription factors in plants and play critical regulatory roles in stress tolerance and the development of plants. In our study, CiWRKY48 was cloned from C. intermedia, analyzed using bioinformatics software, and expressed with a prokaryotic expression system. The results showed that the open reading frame (ORF) of CiWRKY48 was 1158bp, the molecular weight (MW) was 42 kDa, and its subcellular localization was in the nucleus. Additionally, fusion protein was obtained, and confirmed by western blotting. The stress resistance of the pET30a-His-MBP-CiWRKY48 transformed Escherichia coli expression strain was reduced under mannitol and salt treatment, compared with the control. Overall, our findings provided a foundation for uncovering the function of CiWRKY48.

Phase Change Materials Application in Battery Thermal Management System: A Review.

The purpose of a battery thermal management system (BTMS) is to maintain the battery safety and efficient use as well as ensure the battery temperature is within the safe operating range. The traditional air-cooling-based BTMS not only needs extra power, but it could also not meet the demand of new lithium-ion battery (LIB) packs with high energy density, while liquid cooling BTMS requires complex devices to ensure the effect. Therefore, phase change materials (PCMs)-based BTMS is becoming the trend. By using PCMs to absorb heat, the temperature of a battery pack could be kept within the normal operating range for a long time without using any external power. PCMs could greatly improve the heat dissipation efficiency of BTMS by combining with fillers such as expanded graphite (EG) and metal foam for their high thermal conductivity or coordinating with fins. In addition, PCMs could also be applied in construction materials, solar thermal recovery, textiles and other fields. Herein, a comprehensive review of the PCMs applied in thermal storage devices, especially in BTMS, is provided. In this work, the literature concerning current issues have been reviewed and summarized, while the key challenges of PCM application have been pointed out. This review may bring new insights to the PCM application.

Analysis of Metabolite Accumulation Related to Pod Color Variation of Caragana intermedia.

Caragana intermedia, a leguminous shrub widely distributed in cold and arid regions, is rich in secondary metabolites and natural active substances, with high nutritional and medical values. It is interesting that the pods of C. intermedia often show different colors among individual plants. In this study, 10-, 20- and 30-day-old red and green pods of C. intermedia were used to identify and characterize important metabolites associated with pod color. A total 557 metabolites, which could be classified into 21 groups, were detected in the pod extracts using liquid chromatography coupled with ESI-triple quadrupole-linear ion trap mass spectrometer (LC-ESI-MS/MS). Metabolomics analysis revealed significant differences in 15 groups of metabolites between red and green pods, including amino acids, nucleotide derivatives, flavonoids, and phytohormones. Metabolic pathway analysis showed that the shikimic acid and the phytohormone metabolic pathways were extraordinarily active in red pods, and the difference between red and green pods was obvious. Moreover, red pods showed remarkable flavonoids, cytokinins, and auxin accumulation, and the content of total flavonoids and proanthocyanidins in 30-day-old red pods was significantly higher than that in green pods. This metabolic profile contributes to valuable insights into the metabolic regulation mechanism in different color pods.

[Preparation of immobilized Lactobacillus plantarum agent for silage].

Lactic acid bacteria and cellulose degrading bacteria play an important role in fermentation process of silage, because they can prevent the rancidity and increase the nutritive value of silage. But the propagation of lactic acid bacteria will inhibit the activity of cellulose degrading bacteria in the silage fermentation system. This problem can be solved by releasing lactic acid bacteria and cellulose degrading bacteria in different time. Therefore, we immobilized lactic acid bacteria as a microbial agent for sustained release. Firstly, the optimal balling concentration of the composite immobilized carrier and composite immobilized carrier were obtained by immobilization of blank balls and corncob adsorbed Lactobacillus plantarum S1 respectively. The best immobilization condition of L. plantarum S1 was obtained by comparing the immobilized rate and balling effect of two kinds of balls, which were embedded by sodium alginate (SA), CMC-Na and embedded-crosslinked by SA, CMC-Na, polyvinyl alcohol (PVA). The results showed that the best balling concentration was achieved by using 6% PVA+0.4% SA+0.3% CMC-Na for embedding-crosslinking and 1.2% SA+0.5% CMC-Na for direct embedding respectively. In addition, comparing with the mechanical strength and embedding rate of five kinds of immobilization process, the best immobilized process was obtained by adding of the mixture of immobilized carriers (1.2%SA+ 0.5%CMC-Na) and corncob adsorbed L. plantarum S1 slowly into 4% CaCl2 for 24 hours. The corncob adsorption and SA embedding methodology can effectively increase the embedding efficiency of Lactobacillus plantarum S1.

Genome-wide Identification of PP2C Genes and Their Expression Profiling in Response to Drought and Cold Stresses in Medicago truncatula.

Type 2 C protein phosphatases (PP2Cs) represent the major group of protein phosphatases in plants and play important roles in various plant processes. In this study, 94 MtPP2C genes were identified from Medicago truncatula and further phylogenetically classified into 13 subfamilies, as supported by exon-intron organization and conserved motif composition. Collinearity analysis indicated that segmental duplication events played a crucial role in the expansion of MtPP2C gene families in M. truncatula. Furthermore, the expression profiles of MtPP2Cs under different abiotic treatments were analyzed using qRT-PCR. Results showed that these MtPP2Cs genes displayed different expression patterns in response to drought, cold and ABA stress conditions and some of the key stress responsive MtPP2Cs genes have been identified. Our study presents a comprehensive overview of the PP2C gene family in M. truncatula, which will be useful for further functional characterization of MtPP2Cs in plant drought and cold stress responses.

Identification of the WRKY gene family and functional analysis of two genes in Caragana intermedia.

BACKGROUND: WRKY transcription factors, one of the largest families of transcriptional regulators in plants, play important roles in plant development and various stress responses. The WRKYs of Caragana intermedia are still not well characterized, although many WRKYs have been identified in various plant species. RESULTS: We identified 53 CiWRKY genes from C. intermedia transcriptome data, 28 of which exhibited complete open reading frames (ORFs). These CiWRKYs were divided into three groups via phylogenetic analysis according to their WRKY domains and zinc finger motifs. Conserved domain analysis showed that the CiWRKY proteins contain a highly conserved WRKYGQK motif and two variant motifs (WRKYGKK and WKKYEEK). The subcellular localization of CiWRKY26 and CiWRKY28-1 indicated that these two proteins localized exclusively to nuclei, supporting their role as transcription factors. The expression patterns of the 28 CiWRKYs with complete ORFs were examined through quantitative real-time PCR (qRT-PCR) in various tissues and under different abiotic stresses (drought, cold, salt, high-pH and abscisic acid (ABA)). The results showed that each CiWRKY responded to at least one stress treatment. Furthermore, overexpression of CiWRKY75-1 and CiWRKY40-4 in Arabidopsis thaliana suppressed the drought stress tolerance of the plants and delayed leaf senescence, respectively. CONCLUSIONS: Fifty-three CiWRKY genes from the C. intermedia transcriptome were identified and divided into three groups via phylogenetic analysis. The expression patterns of the 28 CiWRKYs under different abiotic stresses suggested that each CiWRKY responded to at least one stress treatment. Overexpression of CiWRKY75-1 and CiWRKY40-4 suppressed the drought stress tolerance of Arabidopsis and delayed leaf senescence, respectively. These results provide a basis for the molecular mechanism through which CiWRKYs mediate stress tolerance.

Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses.

Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes (EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2) were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress.

De novo assembly and transcriptomic profiling of the grazing response in Stipa grandis.

BACKGROUND: Stipa grandis (Poaceae) is one of the dominant species in a typical steppe of the Inner Mongolian Plateau. However, primarily due to heavy grazing, the grasslands have become seriously degraded, and S. grandis has developed a special growth-inhibition phenotype against the stressful habitat. Because of the lack of transcriptomic and genomic information, the understanding of the molecular mechanisms underlying the grazing response of S. grandis has been prohibited. RESULTS: Using the Illumina HiSeq 2000 platform, two libraries prepared from non-grazing (FS) and overgrazing samples (OS) were sequenced. De novo assembly produced 94,674 unigenes, of which 65,047 unigenes had BLAST hits in the National Center for Biotechnology Information (NCBI) non-redundant (nr) database (E-value < 10-5). In total, 47,747, 26,156 and 40,842 unigenes were assigned to the Gene Ontology (GO), Clusters of Orthologous Group (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. A total of 13,221 unigenes showed significant differences in expression under the overgrazing condition, with a threshold false discovery rate ≤ 0.001 and an absolute value of log2Ratio ≥ 1. These differentially expressed genes (DEGs) were assigned to 43,257 GO terms and were significantly enriched in 32 KEGG pathways (q-value ≤ 0.05). The alterations in the wound-, drought- and defense-related genes indicate that stressors have an additive effect on the growth inhibition of this species. CONCLUSIONS: This first large-scale transcriptome study will provide important information for further gene expression and functional genomics studies, and it facilitated our investigation of the molecular mechanisms of the S. grandis grazing response and the associated morphological and physiological characteristics.

[Isolation, identification and characterization of acid-producing strains from psychrotolerant biogas fermentation].

OBJECTIVE: The aim of this study was to screen acid-producing strains from the broth of psychrotolerant biogas fermentation and evaluate the acid-producing character of them. METHODS: Acid-producing strains were isolated by a medium with methyl red at 4 degrees C in Petri dishes and identified by morphology observation and 16S rRNA sequencing. Moreover, the ability of hydrolysis of starch, fermentation of carbohydrates, liquefaction of gelatin and production of catalase were studied. RESULTS: Two acid-producing strains (FJ-8 and FJ-15) were isolated. The result of the 16S rRNA phylogenetic tree shows that FJ-8 and FJ-15 belong to Pseudomonas sp. and Shewanella sp., respectively. Both FJ-8 and FJ-15 could hydrolyze starch, liquidize gelatin and produce catalase. The optimum temperature for acid-producing of FJ-8 and FJ-15 is 15 degrees C and 20 degrees C, respectively. After 10 days cultivation at 4 degrees C, the concentration of acetic acid was 792 mg/L and 966 mg/L of FJ-8 and FJ-15, respectively. CONCLUSION: The selected strains, FJ-8 and FJ-15, have the potential to produce acids at low temperature.

Direct electrochemistry and electrochemical catalysis of immobilized hemoglobin in an ethanol-water mixture.

Hemoglobin (Hb) was immobilized on a glassy carbon electrode (GCE) surface by konjac glucomannan (KGM). KGM hydrogel films on GCE have relatively high stabilities in aqueous-ethanol mixtures. The entrapped hemoglobin undergoes fast direct electron transfer reactions in aqueous-organic solvent mixtures. The peak current is bigger, the peak-to-peak separation smaller and the formal potential observed in the cyclic voltammogram is more negative for Hb-KGM/GCE in ethanol-PBS compared to Hb-KGM/GCE in PBS. The electrochemical properties of the Hb in aqueous-organic solution are almost unchanged from with those observed for the purely aqueous solution, suggesting that water pools in the KGM hydrogel play an important role in preventing changes in conformation and making proteins unreactive with polar organic solvents. The immobilized Hb was able to catalyze the reduction of nitric oxide, peroxides (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, 2-butanone peroxide), and the dehalogenation of haloethanes (hexachloroethane, pentachloroethane, tetrachloroethane, etc.). The stability and reproducibility of the modified electrode meant that it could be used to determine these substances.