Pingping Liu

Colorimetric detection of Hg2+ and Pb2+ based on peroxidase-like activity of graphene oxide–gold nanohybrids

In recent years, considerable efforts have been devoted to the construction of efficient enzyme mimetics, which have significant advantages of simple synthesis and good stability. In this paper, we prepared novel graphene oxide–gold (GO–AuNP) nanohybrids that exhibited fascinating peroxidase-like activity and could catalyze the oxidation of the peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) by H2O2, while simultaneously developing a blue color in an aqueous solution. Furthermore, single-stranded DNA (ssDNA) were able to resist the salt-induced aggregation of GO–AuNP nanohybrids, whereas double-stranded DNA (dsDNA) did not inhibit salt-induced GO–AuNP nanohybrids aggregation. Based on these unique properties, we developed a label-free colorimetric method for the detection of Hg2+ and Pb2+. In the presence of Hg2+ or Pb2+, ssDNA formed a hairpin-like or a quadruplex structure, and these conformational changes facilitated the salt-induced aggregation of GO–AuNP nanohybrids, leaving behind a transparent supernatant after centrifugation. After incubation with TMB and H2O2, the colorimetric signal of the centrifugal supernatant was significantly decreased compared to that in the absence of Hg2+ or Pb2+. The response to Hg2+ and Pb2+ were in the range of 0–50 μM. Finally, this simple and sensitive sensor could be successfully applied for the detection of Hg2+ and Pb2+ in river water.

Friend or foe: differential responses of rice to invasion by mutualistic or pathogenic fungi revealed by RNAseq and metabolite profiling.

The rice endophyte Harpophora oryzae shares a common pathogenic ancestor with the rice blast fungus Magnaporthe oryzae. Direct comparison of the interactions between a single plant species and two closely-related (1) pathogenic and (2) mutualistic fungi species can improve our understanding of the evolution of the interactions between plants and fungi that lead to either mutualistic or pathogenic interactions. Differences in the metabolome and transcriptome of rice in response to challenge by H. or M. oryzae were investigated with GC-MS, RNA-seq, and qRT-PCR. Levels of metabolites of the shikimate and lignin biosynthesis pathways increased continuously in the M. oryzae-challenged rice roots (Mo-roots); these pathways were initially induced, but then suppressed, in the H. oryzae-challenged rice roots (Ho-roots). Compared to control samples, concentrations of sucrose and maltose were reduced in the Ho-roots and Mo-roots. The expression of most genes encoding enzymes involved in glycolysis and the TCA cycle were suppressed in the Ho-roots, but enhanced in the Mo-roots. The suppressed glycolysis in Ho-roots would result in the accumulation of glucose and fructose which was not detected in the Mo-roots. A novel co-evolution pattern of fungi-host interaction is proposed which highlights the importance of plant host in the evolution of fungal symbioses.

Integrated mRNA and microRNA analysis identifies genes and small miRNA molecules associated with transcriptional and post-transcriptional-level responses to both drought stress and re-watering treatment in tobacco

BackgroundDrought stress is one of the most severe problem limited agricultural productivity worldwide. It has been reported that plants response to drought-stress by sophisticated mechanisms at both transcriptional and post-transcriptional levels. However, the precise molecular mechanisms governing the responses of tobacco leaves to drought stress and water status are not well understood. To identify genes and miRNAs involved in drought-stress responses in tobacco, we performed both mRNA and small RNA sequencing on tobacco leaf samples from the following three treatments: untreated-control (CL), drought stress (DL), and re-watering (WL).ResultsIn total, we identified 798 differentially expressed genes (DEGs) between the DL and CL (DL vs. CL) treatments and identified 571 DEGs between the WL and DL (WL vs. DL) treatments. Further analysis revealed 443 overlapping DEGs between the DL vs. CL and WL vs. DL comparisons, and, strikingly, all of these genes exhibited opposing expression trends between these two comparisons, strongly suggesting that these overlapping DEGs are somehow involved in the responses of tobacco leaves to drought stress. Functional annotation analysis showed significant up-regulation of genes annotated to be involved in responses to stimulus and stress, (e.g., late embryogenesis abundant proteins and heat-shock proteins) antioxidant defense (e.g., peroxidases and glutathione S-transferases), down regulation of genes related to the cell cycle pathway, and photosynthesis processes. We also found 69 and 56 transcription factors (TFs) among the DEGs in, respectively, the DL vs. CL and the WL vs. DL comparisons. In addition, small RNA sequencing revealed 63 known microRNAs (miRNA) from 32 families and 368 novel miRNA candidates in tobacco. We also found that five known miRNA families (miR398, miR390, miR162, miR166, and miR168) showed differential regulation under drought conditions. Analysis to identify negative correlations between the differentially expressed miRNAs (DEMs) and DEGs revealed 92 mRNA-miRNA interactions between CL and DL plants, and 32 mRNA-miRNA interactions between DL and WL plants.ConclusionsThis study provides a global view of the transcriptional and the post-transcriptional responses of tobacco under drought stress and re-watering conditions. Our results establish an empirical foundation that should prove valuable for further investigations into the molecular mechanisms through which tobacco, and plants more generally, respond to drought stress at multiple molecular genetic levels.

Graphene Oxide-Based Homogeneous Fluorescence Sensor for Multiplex Determination of Various Targets by a Multifunctional Aptamer

A sensitive and selective fluorescent aptasensor for the determination of metal ions, small molecules, sequence-specific DNA, and proteins was fabricated. The analytical approach was based on target-induced conformational changes of the probe and the self-assembled probe-graphene oxide architecture. The probe employed fluorescence “on/off” switching in a single step in solution. This approach was simple to prepare and had little background with good sensitivity and rapid response. In the absence of targets, the probe adsorbed on the surface of graphene oxide through π–π stacking and quenched fluorescence of the probe. Upon the addition of analyte, the random coil of the probe bonded to the targets, reducing the interaction between the probe and graphene oxide, which disrupted the energy transfer from the probe to graphene oxide and therefore increased the fluorescence. This approach was employed for the determination of Ag+, Hg2+, cysteine, sequence-specific DNA, and thrombin. The results demonstrated that the probe-graphene oxide architecture was an excellent and versatile platform for the determination of multiple analytes.

Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance.

Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

Integrated transcriptomics and metabolomics analysis to characterize cold stress responses in Nicotiana tabacum

BackgroundCB-1 and K326 are closely related tobacco cultivars; however, their cold tolerance capacities are different. K326 is much more cold tolerant than CB-1.ResultsWe studied the transcriptomes and metabolomes of CB-1 and K326 leaf samples treated with cold stress. Totally, we have identified 14,590 differentially expressed genes (DEGs) in CB-1 and 14,605 DEGs in K326; there was also 200 differentially expressed metabolites in CB-1 and 194 in K326. Moreover, there were many overlapping genes (around 50%) that were cold-responsive in both plant cultivars, although there were also many differences in the cold responsive genes between the two cultivars. Importantly, for most of the overlapping cold responsive genes, the extent of the changes in expression were typically much more pronounced in K326 than in CB-1, which may help explain the superior cold tolerance of K326. Similar results were found in the metabolome analysis, particularly with the analysis of primary metabolites, including amino acids, organic acids, and sugars. The large number of specific responsive genes and metabolites highlight the complex regulatory mechanisms associated with cold stress in tobacco. In addition, our work implies that the energy metabolism and hormones may function distinctly between CB-1 and K326.ConclusionsDifferences in gene expression and metabolite levels following cold stress treatment seem likely to have contributed to the observed difference in the cold tolerance phenotype of these two tobacco cultivars.

Downregulation of the lycopene ε-cyclase gene confers tolerance to salt and drought stress in Nicotiana tabacum

Lycopene ε-cyclase (ε-LCY) functions at a branch point of the carotenoid biosynthesis pathway and modulates the ratio of lutein to the β-carotenoids. RNA interference (RNAi) and overexpression (OE) of Ntε-LCY were used to evaluate the physiological roles of ε-LCY in Nicotiana tobacum. In leaves, strong accumulation of β-branch carotenoids and high expression of carotenoid biosynthesis genes resulted from suppression of Ntε-LCY expression. RNAi plants showed enhanced salt and drought tolerance, while overexpression of the Ntε-LCY gene weakened tolerance to salt and drought stress, as compared to control. Further analysis revealed that RNAi plants exhibited less water loss and had lower reactive oxygen species levels than did WT plants after both the salt and drought treatments. Further, higher levels of ABA accumulated in the RNAi lines than in the WT lines under stress conditions. These results suggest that reduced Ntε-LCY expression can improve drought and salinity tolerance in Nicotiana tabacum by enhancing their ROS scavenging ability.

Metabolic changes in primary, secondary, and lipid metabolism in tobacco leaf in response to topping

AbstractAs an important cultivation practice used for flue-cured tobacco, topping affects diverse biological processes in the later stages of development and growth. Some studies have focused on using tobacco genes to reflect the physiological changes caused by topping. However, the complex metabolic shifts in the leaf resulting from topping have not yet been investigated in detail. In this study, a comprehensive metabolic profile of primary, secondary, and lipid metabolism in flue-cured tobacco leaf was generated with use of a multiple platform consisting of gas chromatography–mass spectrometry, capillary electrophoresis–mass spectrometry, and liquid chromatography–mass spectrometry/ultraviolet spectroscopy. A total of 367 metabolites were identified and determined. Both principal component analysis and the number of significantly different metabolites indicated that topping had the greatest influence on the upper leaves. During the early stage of topping, great lipid level variations in the upper leaves were observed, and antioxidant defense metabolites were accumulated. This indicated that the topping activated lipid turnover and the antioxidant defense system. At the mature stage, lower levels of senescence-related metabolites and higher levels of secondary metabolites were found in the topped mature leaves. This implied that topping delayed leaf senescence and promoted secondary metabolite accumulation. This study provides a global view of the metabolic perturbation in response to topping. Graphical abstractMetabolic alterations in tobacco leaf in response to topping using a multiplatform metabolomics

Combining Targeted Metabolites Analysis and Transcriptomics to Reveal Chemical Composition Difference and Underlying Transcriptional Regulation in Maca (Lepidium Meyenii Walp.) Ecotypes

Maca (Lepidium meyenii Walp.) is a traditional Andean crop with great potential for various sanitarian and medical functions, which is attracting increased research attention. The majority of previous Maca studies were focused on biochemistry and pharmacodynamics, while the genetic basis of its unique characteristics lagged due to a lack of genome information. The authors perform gas chromatography-mass spectrometry (GC/MS) analysis in the hypocotyls of three Maca ecotypes and identify 79 compounds. Among them, 62 compounds have distinct profiles among Maca ecotypes. To reveal the underlying regulatory mechanism of the chemical composition differences, de novo transcriptome sequencing is performed and the transcription profiles of three Maca ecotypes are comparatively analyzed. Functional analysis indicates several key pathways, including “starch and sucrose metabolism,” “phenylpropanoid biosynthesis,” “phenylalanine metabolism” and “plant-pathogen interaction,” are involved in regulating the chemical compositions of Maca. Combining metabolomics and transcriptomics analysis indicates transcription factors such as MYB and WRKY and mediators such as protein kinase and bifunctional inhibitors might be critical regulators of chemical composition in Maca. The transcriptome reference genome and differentially expressed genes (DEGs) obtained in this study might serve as an initial step to illustrate the genetic differences in nutrient component, secondary metabolites content, medicinal function and stress resistance in Maca.

Identification and analysis of the chloride channel gene family members in tobacco (Nicotiana tabacum).

The chloride channel (CLC) protein family, which includes both chloride (Cl-) channels and chloride/proton (Cl-/H+) antiporters, is present in all domains of life, from prokaryotes to eukaryotes. However, there are no reported studies about this gene family in tobacco, an economically important global crop plant. In this study, we identified seventeen CLC genes in the genome of Nicotiana tabacum. A multiple sequence alignment showed that all of the predicted proteins shared a high sequence similarity and had a highly conserved GKxGPxxH motif. A gene structure analysis revealed that the NtCLC genes had highly divergent intron-exon patterns. A phylogenetic and conserved motif analysis revealed that the NtCLC family was divided into two clades, in a manner similar to other plants. We also evaluated the expression patterns of these NtCLC genes in different tissues and in plants treated with salt stress. The NtCLC genes had highly variable expression patterns, for example, the largely stem- and bud-specific expression patterns of NtCLC6 and NtCLC8, respectively. Salt stress treatment (300 mM NaCl) induced the expression of NtCLC2, NtCLC3, and NtCLC12, suggesting that these genes might play a role in tobacco responses to salt stress. Furthermore, the concentration of Cl- in the NtCLC2- and NtCLC13-silenced plants showed an obvious lower and higher level, respectively, than the control plants. Thus, we indicated that NtCLC2 or NtCLC13 might play an important role in chloride transport or metabolism in tobacco. Together, these findings establish an empirical foundation for the further functional characterization of the NtCLC genes in tobacco.