Yuan Song

The tandem affinity purification method: An efficient system for protein complex purification and protein interaction identification

Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method.

Chilling- and Freezing- Induced Alterations in Cytosine Methylation and Its Association with the Cold Tolerance of an Alpine Subnival Plant, Chorispora bungeana

Chilling (0–18°C) and freezing (<0°C) are two distinct types of cold stresses. Epigenetic regulation can play an important role in plant adaptation to abiotic stresses. However, it is not yet clear whether and how epigenetic modification (i.e., DNA methylation) mediates the adaptation to cold stresses in nature (e.g., in alpine regions). Especially, whether the adaptation to chilling and freezing is involved in differential epigenetic regulations in plants is largely unknown. Chorispora bungeana is an alpine subnival plant that is distributed in the freeze-thaw tundra in Asia, where chilling and freezing frequently fluctuate daily (24 h). To disentangle how C. bungeana copes with these intricate cold stresses through epigenetic modifications, plants of C. bungeana were treated at 4°C (chilling) and -4°C (freezing) over five periods of time (0–24 h). Methylation-sensitive amplified fragment-length polymorphism markers were used to investigate the variation in DNA methylation of C. bungeana in response to chilling and freezing. It was found that the alterations in DNA methylation of C. bungeana largely occurred over the period of chilling and freezing. Moreover, chilling and freezing appeared to gradually induce distinct DNA methylation variations, as the treatment went on (e.g., after 12 h). Forty-three cold-induced polymorphic fragments were randomly selected and further analyzed, and three of the cloned fragments were homologous to genes encoding alcohol dehydrogenase, UDP-glucosyltransferase and polygalacturonase-inhibiting protein. These candidate genes verified the existence of different expressive patterns between chilling and freezing. Our results showed that C. bungeana responded to cold stresses rapidly through the alterations of DNA methylation, and that chilling and freezing induced different DNA methylation changes. Therefore, we conclude that epigenetic modifications can potentially serve as a rapid and flexible mechanism for C. bungeana to adapt to the intricate cold stresses in the alpine areas.

Metabolite Profiling of adh1 Mutant Response to Cold Stress in Arabidopsis

As a result of global warming, vegetation suffers from repeated freeze-thaw cycles caused by more frequent short-term low temperatures induced by hail, snow, or night frost. Therefore, short-term freezing stress of plants should be investigated particularly in light of the current climatic conditions. Alcohol dehydrogenase (ADH) plays a central role in the metabolism of alcohols and aldehydes and it is a key enzyme in anaerobic fermentation. ADH1 responds to plant growth and environmental stress; however, the function of ADH1 in the response to short-term freezing stress remains unknown. Using real-time quantitative fluorescence PCR, the expression level of ADH1 was analyzed at low temperature (4°C). The lethal temperature was calculated based on the electrolyte leakage tests for both ADH1 deletion mutants (adh1) and wild type (WT) plants. To further investigate the relationship between ADH1 and cold tolerance in plants, low-Mr polar metabolite analyses of Arabidopsis adh1 and WT were performed at cold temperatures using gas chromatography-mass spectrometry. This investigation focused on freezing treatments (cold acclimation group: −6°C for 2 h with prior 4°C for 7 d, cold shock group: −6°C for 2 h without cold acclimation) and recovery (23°C for 24 h) with respect to seedling growth at optimum temperature. The experimental results revealed a significant increase in ADH1 expression during low temperature treatment (4°C) and at a higher lethal temperature in adh1 compared to that in the WT. Retention time indices and specific mass fragments were used to monitor 263 variables and annotate 78 identified metabolites. From these analyses, differences in the degree of metabolite accumulation between adh1 and WT were detected, including soluble sugars (e.g., sucrose) and amino acids (e.g., asparagine). In addition, the correlation-based network analysis highlighted some metabolites, e.g., melibiose, fumaric acid, succinic acid, glycolic acid, and xylose, which enhanced connectedness in adh1 network under cold chock. When considered collectively, the results showed that adh1 possessed a metabolic response to freezing stress and ADH1 played an important role in the cold stress response of a plant. These results expands our understanding of the short-term freeze response of ADH1 in plants.

Using the properties of soil to speed up the start-up process, enhance process stability, and improve the methane content and yield of solid-state anaerobic digestion of alkaline-pretreated poplar processing residues

BackgroundSolid-state anaerobic digestion (SS-AD) was initially adopted for the treatment of municipal solid waste. Recently, SS-AD has been increasingly applied to treat lignocellulosic biomass, such as agricultural and forestry residues. However, studies on the SS-AD process are few. In this study, the process performance and methane yield from SS-AD of alkaline-pretreated poplar processing residues (PPRs) were investigated using the properties of soil, such as buffering capacity and nutritional requirements.ResultsThe results showed that the lignocellulosic structures of the poplar sample were effectively changed by NaOH pretreatment, as indicated by scanning electron microscopy and Fourier transform infrared spectra analysis. The start-up was markedly hastened, and the process stability was enhanced. After NaOH pretreatment, the maximum methane yield (96.1 L/kg volatile solids (VS)) was obtained under a poplar processing residues-to-soil sample (P-to-S) ratio of 2.5:1, which was 29.9% and 36.1% higher than that of PPRs (74.0 L/kg VS) and that of experiments without NaOH pretreatment (70.6 L/kg VS), respectively. During steady state, the increase in the methane content of the experiment with a P-to-S ratio of 2.5:1 was 4.4 to 50.9% higher than that of the PPRs. Degradation of total solids and volatile solids ranged from 19.3 to 33.0% and from 34.9 to 45.9%, respectively. The maximum reductions of cellulose and hemicellulose were 52.6% and 42.9%, respectively, which were in accordance with the maximal methane yield. T80 for the maximum methane yield for the experiments with NaOH pretreatment was 11.1% shorter than that for the PPRs.ConclusionsPretreatment with NaOH and addition of soil led to a significant improvement in the process performance and the methane yield of SS-AD of PPRs. The changes in lignocellulosic structures induced by NaOH pretreatment led to an increase in methane yield. For the purpose of practical applications, SS-AD with soil addition is a convenient, economical, and practical technique.

Trichostatin A and 5-Aza-2′-Deoxycytidine influence the expression of cold-induced genes in Arabidopsis

ABSTRACT The expression of cold-induced genes is critical for plants to survive under freezing stress. However, the underlying mechanisms for the decision of when, where, and which genes to express are unclear when a plant meets a sudden temperature drop. Previous studies have demonstrated epigenetics to play a central role in the regulation of gene expression in plant responses to environmental stress. DNA methylation and histone deacetylation are the two most important epigenetic modifications. This study was conducted to investigate the effects of inhibiting DNA methylation and histone deacetylation on gene expression, and to explore the potential role of epigenetics in plant responses to cold stress. The results revealed that histone deacetylase inhibitors (trichostatin A) and DNA methylation inhibitors (5-Aza-2′-deoxycytosine) treatment enhanced cold tolerance. DNA microarray analysis and the gene ontology method revealed 76 cold-induced differently expressed genes in Arabidopsis thaliana seedlings that were treated to 0°C for 24 h following Trichostatin A and 5-Aza-2′-Deoxycytidine. Furthermore, analyses of metabolic pathways and transcription factors of 3305 differentially expressed genes were performed. Each four metabolic pathways were significantly affected (p < 0.01) by Trichostatin A and 5-Aza-2′-Deoxycytidine. Finally, 10 genes were randomly selected and verified via qPCR analysis. Our study indicated that Trichostatin A and 5-Aza-2′-Deoxycytidine can improve the plant cold resistance and influence the expression of the cold-induced gene in A. thaliana. This result will advance our understanding of plant freezing responses and may provide a helpful strategy for cold tolerance improvement in crops.

Differential expression by chromatin modifications of alcohol dehydrogenase 1 of Chorispora bungeana in cold stress

Epigenetic modifications regulate plant genes to cope with a variety of environmental stresses. Chorispora bungeana is an alpine subnival plant with strong tolerance to multiple abiotic stresses, especially cold stress. In this study, we characterized the alcohol dehydrogenase 1 gene from Chorispora bungeana, CbADH1, that is up-regulated in cold conditions. Overexpression of CbADH1 in Arabidopsis thaliana improved cold tolerance, as indicated by a decreased lethal temperature (LT50). Chromatin immunoprecipitation assays showed that histone H3 is removed from the promoter region and the middle-coding region of the gene. H3K9 acetylation and H3K4 trimethylation increased throughout the gene and in the proximal promoter region, respectively. Moreover, increased Ser5P and Ser2P polymerase II accumulation further indicated changes in the transcription initiation and elongation of CbADH1 were due to the cold stress. Taken together, our results suggested that CbADH1 is highly expressed during cold stress, and is regulated by epigenetic modifications. This study expands our understanding of the regulation of gene expression by epigenetic modifications in response to environmental cues.