Chemical modification of transcripts with 5′ caps occurs in all organisms. Here, we report a systems-level mass spectrometry-based technique, CapQuant, for quantitative analysis of an organism's cap epitranscriptome. The method was piloted with 21 canonical caps—m7GpppN, m7GpppNm, GpppN, GpppNm, and m2,2,7GpppG—and 5 ‘metabolite’ caps—NAD, FAD, UDP-Glc, UDP-GlcNAc, and dpCoA. Applying CapQuant to RNA from purified dengue virus, Escherichia coli, yeast, mouse tissues, and human cells, we discovered new cap structures in humans and mice (FAD, UDP-Glc, UDP-GlcNAc, and m7Gpppm6A), cell- and tissue-specific variations in cap methylation, and high proportions of caps lacking 2′-O-methylation (m7Gpppm6A in mammals, m7GpppA in dengue virus). While substantial Dimroth-induced loss of m1A and m1Am arose with specific RNA processing conditions, human lymphoblast cells showed no detectable m1A or m1Am in caps. CapQuant accurately captured the preference for purine nucleotides at eukaryotic transcription start sites and the correlation between metabolite levels and metabolite caps.
Living biological systems, ranging from single cells to whole organisms, can sense, process information, and actuate in response to changing environmental conditions. Inspired by living biological systems, engineered living cells and nonliving matrices are brought together, which gives rise to the technology of engineered living materials. By designing the functionalities of living cells and the structures of nonliving matrices, engineered living materials can be created to detect variability in the surrounding environment and to adjust their functions accordingly, thereby enabling applications in health monitoring, disease treatment, and environmental remediation. Hydrogels, a class of soft, wet, and biocompatible materials, have been widely used as matrices for engineered living cells, leading to the nascent field of engineered living hydrogels. Here, the interactions between hydrogel matrices and engineered living cells are described, focusing on how hydrogels influence cell behaviors and how cells affect hydrogel properties. The interactions between engineered living hydrogels and their environments, and how these interactions enable versatile applications, are also discussed. Finally, current challenges facing the field of engineered living hydrogels for their applications in clinical and environmental settings are highlighted.
Very little is known about the adaptation mechanism of Chenopodiaceae Halogeton glomeratus, a succulent annual halophyte, under saline conditions. In this study, we investigated the morphological and physiological adaptation mechanisms of seedlings exposed to different concentrations of NaCl treatment for 21 d. Our results revealed that H. glomeratus has a robust ability to tolerate salt; its optimal growth occurs under approximately 100 mm NaCl conditions. Salt crystals were deposited in water-storage tissue under saline conditions. We speculate that osmotic adjustment may be the primary mechanism of salt tolerance in H. glomeratus, which transports toxic ions such as sodium into specific salt-storage cells and compartmentalizes them in large vacuoles to maintain the water content of tissues and the succulence of the leaves. To investigate the molecular response mechanisms to salt stress in H. glomeratus, we conducted a comparative proteomic analysis of seedling leaves that had been exposed to 200 mm NaCl for 24 h, 72 h and 7 d. Forty-nine protein spots, exhibiting significant changes in abundance after stress, were identified using matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS/MS) and similarity searches across EST database of H. glomeratus. These stress-responsive proteins were categorized into nine functional groups, such as photosynthesis, carbohydrate and energy metabolism, and stress and defence response.
Edible plant oil (EPO) is an indispensable nutritional resource for human health. Various cultivars of oil-bearing plants are grown worldwide, and the chemical compositions of different plant oils are diverse. The extremely complex components in oils lead to diverse standards for evaluating the quality and safety of different EPOs. The environment poses great challenges to the EPO safety and quality during the entire industrial chain, including plant cultivation, harvesting, oil processing, and storage. Environmental risk factors include heavy metal or pesticide residue pollution, insect or harmful microbial infestation, and rancidity. Here, the diverse components in oil and various oil-producing processes are discussed, including plant species, oil yield, and composition complexity, environmental factors that degrade oil quality. Additionally, we propose a whole-industrialchain monitoring system instead of current single-link-monitoring approach by monitoring and tracking the quality and safety of EPOs during the entire process of plant cultivation, raw materials harvest, oil process, and EPOs storage. This will provide guidance for monitoring the quality and safety of EPOs, which were challenged by the deteriorating environment.
A lack of phosphorus (P) in plants can severely constrain growth and development. Barley, one of the earliest domesticated crops, is extensively planted in poor soil around the world. To date, the molecular mechanisms of enduring low phosphorus, at the transcriptional level, in barley are still unclear. In the present study, two different barley genotypes (GN121 and GN42)—with contrasting phosphorus efficiency—were used to reveal adaptations to low phosphorus stress, at three time points, at the morphological, physiological, biochemical, and transcriptome level. GN121 growth was less affected by phosphorus starvation and recovery than that of GN42. The biomass and inorganic phosphorus concentration of GN121 and GN42 declined under the low phosphorus-induced stress and increased after recovery with normal phosphorus. However, the range of these parameters was higher in GN42 than in GN121. Subsequently, a more complete genome annotation was obtained by correcting with the data sequenced on Illumina HiSeq X 10 and PacBio RSII SMRT platform. A total of 6,182 and 5,270 differentially expressed genes (DEGs) were identified in GN121 and GN42, respectively. The majority of these DEGs were involved in phosphorus metabolism such as phospholipid degradation, hydrolysis of phosphoric enzymes, sucrose synthesis, phosphorylation/dephosphorylation and post-transcriptional regulation; expression of these genes was significantly different between GN121 and GN42. Specifically, six and seven DEGs were annotated as phosphorus transporters in roots and leaves, respectively. Furthermore, a putative model was constructed relying on key metabolic pathways related to phosphorus to illustrate the higher phosphorus efficiency of GN121 compared to GN42 under low phosphorus conditions. Results from this study provide a multi-transcriptome database and candidate genes for further study on phosphorus use efficiency (PUE).
6 7 Chemical modification of transcripts with 5' caps occurs in all organisms. Here we report a 8 systems-level mass spectrometry-based technique, CapQuant, for quantitative analysis of the 9 cap epitranscriptome in any organism. The method was piloted with 21 canonical caps -10 m 7 GpppN, m 7 GpppNm, GpppN, GpppNm, and m 2,2,7 GpppG -and 5 "metabolite" caps -NAD, 11 FAD, UDP-Glc, UDP-GlcNAc, and dpCoA. Applying CapQuant to RNA from purified dengue 12 virus, Escherichia coli, yeast, mice, and humans, we discovered four new cap structures in 13 humans and mice (FAD, UDP-Glc, UDP-GlcNAc, and m 7 Gpppm 6 A), cell-and tissue-specific 14 variations in cap methylation, and surprisingly high proportions of caps lacking 2'-O-methylation, 15 such as m 7 Gpppm 6 A in mammals and m 7 GpppA in dengue virus, and we did not detect cap 16 m 1 A/m 1 Am in humans. CapQuant accurately captured the preference for purine nucleotides at 17 eukaryotic transcription start sites and the correlation between metabolite levels and metabolite 18 caps. The mystery around cap m 1 A/m 1 Am analysis remains unresolved. 19 20 21 cap structure involves -phosphate methylation of unprocessed 5'-triphosphate (mPPPN) on 1 small RNAs such as mammalian U6 and 7SK, mouse B2, and plant U3 RNAs (7). 2 A variety of non-canonical caps involving nucleotide metabolites (Figure 1A) have also recently 3 been described (8,9). For example, nicotinamide adenine dinucleotide (NAD) and coenzyme A 4 (CoA) were found as cap-like structures in bacterial small RNAs (10) and the NAD cap was also 5 found in yeast and human mRNA and non-coding RNAs (11). Julius and Yuzenkova expanded 6 the potential repertoire of caps by demonstrating that a variety of nucleotide metabolites could 7 initiate transcription by bacterial RNA polymerase (RNA Pol) in vitro, including flavin adenine 8 dinucleotide (FAD), uridine diphosphate glucose (UDP-Glc), and uridine diphosphate N-9 acetylglucosamine (UDP-GlcNAc) (9). They also showed that capping with NAD and UDP 10 analogs by bacterial RNA Pol is promoter-specific and stimulates promoter escape (9), 11 suggesting a role for metabolite caps in regulating gene expression. For example, the NAD cap 12 has been shown to influence RNA stability and turnover, and is a substrate for decapping 13 enzymes (11). However, the lack of sensitive and specific analytical methods has hindered the 14 systematic study of the cap landscape dynamics in cells. 15Analysis of RNA cap structures has traditionally relied on radioisotope labeling and enzymatic 16 hydrolysis, followed by thin-layer and other types of chromatography to resolve cap structures 17 (12)(13)(14). While sensitive, the radiolabeling approach lacks specificity (12) and has the potential 18 to create cellular toxicity artifacts (15,16). While two-dimensional electrophoresis (14) allows 19 multiple caps analysis, it (i) lacks specificity for identifying intact cap structures, (ii) is limited to 20 NpppN caps, (iii) does not provide absolute quantification, and (iv) is semi-quantitative at b...
BackgroundHalogeton glomeratus (H. glomeratus) is an extreme halophyte that is widely distributed in arid regions, including foothills, the Gobi desert of northwest China, and the marginal loess of Central Asia. However, research on the salt-tolerant mechanisms and genes of this species are limited because of a lack of genomic sequences. In the present study, the transcriptome of H. glomeratus was analyzed using next-generation sequencing technology to identify genes involved in salt tolerance and better understand mechanisms of salt response in the halophyte H. glomeratus.ResultsIllumina RNA-sequencing was performed in five sequencing libraries that were prepared from samples treated with 200 mM NaCl for 6, 12, 24, and 72 h and a control sample to investigate changes in the H. glomeratus transcriptome in response to salt stress. The de novo assembly of five transcriptomes identified 50,267 transcripts. Among these transcripts, 31,496 (62.66%) were annotated, including 44 Gene Ontology (GO) terms and 128 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Compared with transcriptomes from the control and NaCl-treated samples, there were 2,223, 5,643, 7,510 and 10,908 genes that were differentially expressed after exposure to NaCl for 6, 12, 24, and 72 h, respectively. One hundred and eighteen salt-induced genes were common to at least two stages of salt stress, and 291 up-regulated genes were common to various stages of salt stress. Numerous genes that are related to ion transport, reactive oxygen species scavenging, energy metabolism, hormone-response pathways, and responses to biotic and abiotic stress appear to play a significant role in adaptation to salinity conditions in this species. The detection of expression patterns of 18 salt-induced genes by quantitative real-time polymerase chain reaction were basically consistent with their changes in transcript abundance determined by RNA sequencing.ConclusionsOur findings provide a genomic sequence resource for functional genetic assignments of an extreme halophyte, H. glomeratus. We believe that the transcriptome datasets will help elucidate the genetic basis of this species’ response to a salt environment and develop stress-tolerant crops based on favorable wild genetic resources.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1373-z) contains supplementary material, which is available to authorized users.
Soil salinity severely threatens land use capability and crop yields worldwide. An analysis of the molecular mechanisms of salt tolerance in halophytes will contribute to the development of salt-tolerant crops. In this study, a combination of physiological characteristics and iTRAQ-based proteomic approaches was conducted to investigate the molecular mechanisms underlying the salt response of suspension cell cultures of halophytic Halogeton glomeratus. These cells showed halophytic growth responses comparable to those of the whole plant. In total, 97 up-regulated proteins and 192 down-regulated proteins were identified as common to both 200 and 400 mM NaCl concentration treatments. Such salinity responsive proteins were mainly involved in energy, carbohydrate metabolism, stress defense, protein metabolism, signal transduction, cell growth, and cytoskeleton metabolism. Effective regulatory protein expression related to energy, stress defense, and carbohydrate metabolism play important roles in the salt-tolerance of H. glomeratus suspension cell cultures. However, known proteins regulating Na+ efflux from the cytoplasm and its compartmentalization into the vacuole did not change significantly under salinity stress suggesting our existing knowledge concerning Na+ extrusion and compartmentalization in halophytes needs to be evaluated further. Such data are discussed in the context of our current understandings of the mechanisms involved in the salinity response of the halophyte, H. glomeratus.
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