Label-free fluorescent sensor for sensitive detection of ctDNA based on water stabilized CsPbBr3 nanosheet.

The detection of circulating tumor DNA (ctDNA), as a practical liquid biopsy technique, was of great significance for the study of cancer diagnosis and prognosis. However, reported methods for detection ctDNA still have some limitations, such as tedious process and high cost. In this study, CsPbBr3 nanosheet (CsPbBr3 NS) with high water stability was prepared by etching, and its fluorescence intensity could be stably stored for 1 year. The Ti3C2Tx possessed high quenching efficiency for CsPbBr3 NS and the HOMO-LUMO orbital study revealed that the PET mechanism was responsible for fluorescence quenching. And the Ti3C2Tx showed stronger affinity towards single-stranded DNA (ssDNA), as compared with double-stranded DNA (dsDNA). The probe ssDNA could be adsorbed on the surface of Ti3C2Tx through π-π stacking. After the targets were recognized by probe ssDNA to form dsDNA, its affinity with Ti3C2Tx decreased and the active site of Ti3C2Tx recovered, causing a high quenching efficiency on CsPbBr3 NS. Based on this, a label-free fluorescent biosensor was designed for the sensitive detection of ctDNA (EGFR 19 Dels for non-small cell lung cancer, NSCLC). Under the optimal experimental conditions, this biosensor exhibited a detection limit of 180 fM and a linear range of 50 pM-350 pM with amplification of magnetic beads through strand displacement reaction. In addition, this sensor was applied to the detection of ctDNA in serum samples and cells lysates. This method for ctDNA detection was expected to have great potential for biomarker detection in the field of liquid biopsy.

Hydrogen sulfide-induced chilling resistance in peach fruit is performed via sustaining the homeostasis of ROS and RNS.

Hydrogen sulfide (H2S) application impacts on endogenous H2S, reactive oxygen species (ROS), and reactive nitrogen species (RNS) in cold-stored peach fruit were investigated. 20 µL L-1 H2S (NaHS as the donor) efficiently retarded the quality deterioration arising from chilling injury (CI), triggering endogenous H2S production, while enhancing antioxidant systems and ROS generation (NADPH oxidative enzyme). H2S promoted nitric oxide (NO) correlated with the S-nitrosoglutathione reductase (GSNOR)-mediated GSNO reduction, while suppressing the peroxynitrite anion content. As the pivotal coenzyme of ROS and RNS, nicotinamide adenine dinucleotide phosphate (NADPH) levels were elevated by H2S during late-stage storage via the tricarboxylic acid cycle, where reduced NADP-isocitrate dehydrogenase (NADP-ICDH) activity and gene expression. Structural analysis of peach NADP-ICDH (UniProtKB M5WXP5) deduced that Cys79 and Tyr396 are the likeliest targets for S-nitrosylation and nitration, respectively. These results indicate that H2S counteracts the disorders of ROS and RNS to ameliorate CI of peach fruit.

Exogenous 24-epibrassinolide alleviates chilling injury in peach fruit through modulating PpGATA12-mediated sucrose and energy metabolisms.

24-Epibrassinolide (EBR) may act as a modulator for chilling injury in peach fruit during cold storage. In this study, we screened a EBR-induced GATA-type zinc finger protein PpGATA12. The objective of this study was to investigate the potential roles of EBR treatment and transcriptional regulation of PpGATA12 in regulating chilling resistance of peaches. In the current study, we found that EBR treatment promoted the activities and transcriptions of energy and sucrose metabolism-related enzymes, maintained higher ATP content and energy status, improved the accumulation of sucrose and hexose. Furthermore, molecular biology assays suggested that PpGATA12 up-regulated transcriptions of sucrose metabolism-related genes including PpSS and PpNI, and energy metabolism-related genes including PpCCO, PpSDH and PpH+-ATPase. These results provided a new insight that the enhancement of chilling resistance in peach fruit by EBR treatment might be closely related to the regulatory role of PpGATA12 on sucrose and energy metabolisms.

Multi-omics analyses reveal stone cell distribution pattern in pear fruit.

Stone cells are the brachysclereid cells in pear (Pyrus) fruit, consisting almost entirely of lignified secondary cell walls. They are distributed mainly near the fruit core and spread radially in the whole fruit. However, the development of stone cells has not been comprehensively characterized, and little is known about the regulation of stone cell formation at the transcriptomic, proteomic, and metabolomic levels. In the present study, we performed phenomic analysis on the stone cells and their associated vascular bundles distributed near the fruit cores. Transcriptomic, proteomic, and metabolomic analyses revealed a significant positive regulation of biological processes which contribute to the lignification and lignin deposition in stone cells near the fruit core, including sucrose metabolism and phenylalanine, tyrosine, tryptophan, and phenylalanine biosynthesis. We found many metabolites generated from the phenylpropanoid pathway contributing to the cell wall formation of stone cells near the fruit core. Furthermore, we identified a key transcription factor, PbbZIP48, which was highly expressed near the fruit core and was shown to regulate lignin biosynthesis in stone cells. In conclusion, the present study provides insight into the mechanism of lignified stone cell formation near the pear fruit core at multiple levels.

CaCl2 mitigates chilling injury in loquat fruit via the CAMTA5-mediated transcriptional repression of membrane lipid degradation genes.

Loquat fruit is vulnerable to chilling injury (CI) under long-term low temperature storage. The application of calciumchloride (CaCl2) can alleviate CI in loquat fruit, however, the molecular mechanisms remain unclear. In this study, the loquat fruit were immersed in 1 % CaCl2 solution for 10 min and then stored at 1 ± 1 ℃ for 35 d. The results showed that CaCl2 treatment suppressed the increases in internal browning index, electrolyte leakage, and malonaldehyde content in loquat fruit under chilling stress. The internal browning index in CaCl2-treated loquat fruit was 33.34 % lower than that in the control at the end of storage. Meanwhile, CaCl2 treatment delayed the phospholipids degradation, and retained high level of unsaturated/saturated fatty acid ratio, which was 15.21 % higher than that in the control fruit at the end of storage. The CaCl2 treatment also decreased the enzymatic activities and gene expressions of phospholipase D (PLD), phospholipase C (PLC), and lipoxygenase (LOX) in loquat fruit during cold storage. Moreover, a calmodulin-binding transcription activator (CAMTA) transcription factor (TF), EjCAMTA5, was cloned from loquat fruit. The EjCAMTA5 expression was up-regulated by cold stress and CaCl2 treatment. Further investigation revealed that EjCAMTA5 directly bound to the promoters of EjPLC6-like and EjLOX5 to repress their transcription. Taken together, these findings implied that CaCl2 application could activate the EjCAMTA5-mediated transcriptional repression of EjPLC6-like and EjLOX5 genes, which contributed to delaying the degradation of membrane lipid and maintaining the integrity of cell membrane, thereby inhibiting the occurrence of browning in loquat fruit under chilling stress.

Genome-wide identification of the CDPK gene family reveals the CDPK-RBOH pathway potential involved in improving chilling tolerance in peach fruit.

Calcium-dependent protein kinase (CDPK), as an essential calcium receptor, plays a major role in the perception and decoding of intracellular calcium signaling in plant development and stress responses. Here, the CDPK gene family was analyzed at the genome-wide level in peach. This study identified 17 PpCDPK gene members from the peach genome, and systematically investigated phylogenetic relationships, conserved motifs, exon-intron structures, chromosome distribution, and cis-acting elements of each PpCDPK gene using bioinformatics. Furthermore, the expression levels of most PpCDPK genes were significantly changed under the CaCl2, EBR, GB, cold shock, hot water treatments, and various temperatures in the cold storage of peach fruits. RNA-seq data showed that PpCDPK2, PpCDPK7, PpCDPK10, and PpCDPK13 were related to postharvest chilling stress in peach. The interaction network of PpCDPK7 protein showed that the interaction between PpCDPK7 and PpRBOH may be the intersectional point of Ca2+ and ROS signal transmission during cold storage of peach fruits. These systematic analyses are helpful to further characterize the regulation of PpCDPKs and CDPK-RBOH mediated signal cascades in postharvest chilling injury in peach fruit.

PbMC1a/1b regulates lignification during stone cell development in pear (Pyrus bretschneideri) fruit.

Programmed cell death (PCD) and secondary cell wall (SCW) thickening in pear fruit are accompanied by the deposition of cellulose and lignin to form stone cells. Metacaspase is an important protease for development, tissue renewal and PCD. The understanding of the molecular mechanism whereby pear (Pyrus) metacaspase promotes PCD and cell wall lignification is still limited. In this study, the Metacaspases gene family (PbMCs) from P. bretschneideri was identified. PbMC1a/1b was associated with lignin deposition and stone cell formation by physiological data, semiquantitative real-time polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR). Relative to wild-type (WT) Arabidopsis, the overexpression of PbMC1a/1b increased lignin deposition and delayed growth, thickened the cell walls of vessels, xylary fibers and interfascicular fibers, and increased the expression of lignin biosynthetic genes. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and GST pull-down assays indicated that the PbMC1a/1b protein physically interacted with PbRD21. Simultaneously, the transient expression of PbMC1a/1b and PbRD21 led to significant changes in the expression of genes and lignin contents in pear fruits and flesh calli. These results indicate that PbMC1a/1b plays an important role in cell wall lignification, possibly by interacting with PbRD21 to increase the mRNA levels of some lignin synthesis-associated genes and promote the formation of stone cells in pear fruit.

The ß-amylase PbrBAM3 from pear (Pyrus betulaefolia) regulates soluble sugar accumulation and ROS homeostasis in response to cold stress.

ß-Amylase (BAM) is involved in sugar metabolism, but the role of BAM genes in cold tolerance remains poorly understood. Here, we report the identification and functional characterization of the chloroplast-localized BAM-encoding gene PbrBAM3 isolated from Pyrus betulaefolia. The transcript levels of PbrBAM3 were up-regulated under cold, dehydration and ABA, but repressed by maltose. Overexpression of PbrBAM3 in tobacco (Nicotiana tabacum) and pear (P. ussuriensis) conferred increased BAM activity, promoted starch degradation after chilling treatments and enhanced tolerance to cold. Under the chilling stress, the transgenic tobacco and P. ussuriensis exhibited lessened reactive oxygen species (ROS) generation, higher levels of antioxidant enzymes activity, and greater accumulation of soluble sugars (specially maltose) than the corresponding wild type plants. Taken together, these results demonstrate that PbrBAM3 plays an important role in cold tolerance, at least in part, by raising the levels of soluble sugars capable of acting as osmolytes or antioxidants.

Genome-wide analyses and expression patterns under abiotic stress of NAC transcription factors in white pear (Pyrus bretschneideri).

BACKGROUND: Although the genome of Chinese white pear ('Dangshansuli') has been released, little is known about the functions, evolutionary history and expression patterns of NAC families in this species to date. RESULTS: In this study, we identified a total of 183 NAC transcription factors (TFs) in the pear genome, among which 146 pear NAC (PbNAC) members were mapped onto 16 chromosomes, and 37 PbNAC genes were located on scaffold contigs. No PbNAC genes were mapped to chromosome 2. Based on gene structure, protein motif analysis, and topology of the phylogenetic tree, the pear PbNAC family was classified into 33 groups. By comparing and analyzing the unique NAC subgroups in Rosaceae, we identified 19 NAC subgroups specific to pear. We also found that whole-genome duplication (WGD)/segmental duplication played critical roles in the expansion of the NAC family in pear, such as the 83 PbNAC duplicated gene pairs dated back to the two WGD events. Further, we found that purifying selection was the primary force driving the evolution of PbNAC family genes. Next, we used transcriptomic data to study responses to drought and cold stresses in pear, and we found that genes in groups C2f, C72b, and C100a were related to drought and cold stress response. CONCLUSIONS: Through the phylogenetic, evolutionary, and expression analyses of the NAC gene family in Chinese white pear, we indentified 11 PbNAC TFs associated with abiotic stress in pear.

A WRKY transcription factor PbrWRKY53 from Pyrus betulaefolia is involved in drought tolerance and AsA accumulation.

WRKY comprises a large family of transcription factors in plants, but most WRKY members are still poorly understood. In this study, we report the identification and functional characterization of PbrWRKY53 isolated from Pyrus betulaefolia. PbrWRKY53 was greatly up-regulated by drought and abscisic acid, but slightly induced by salt and cold. Subcellar localization analyses showed that PbrWRKY53 was located in the nucleus. Ectopic expression of PbrWRKY53 in tobacco and Pyrus ussuriensis conferred enhanced tolerance to drought stress. The transgenic plants exhibited better water status, less reactive oxygen species generation and higher levels of antioxidant enzyme activities and metabolites than the wild type. In addition, overexpression of PbrWRKY53 in transgenic tobacco resulted in enhanced expression level of PbrNCED1, and led to the increase in larger amount of vitamin C accumulation in comparison to WT. Knock-down of PbrWRKY53 in P. ussuriensis down-regulated PbrNCED1 abundance, accompanied by compromised drought tolerance. Yeast one-hybrid assay, EMSA and transient expression analysis demonstrated that PbrWRKY53 could bind to the W-box element in the promoter region of PbrNCED1. Taken together, these results demonstrated that PbrWRKY53 plays a positive role in drought tolerance, which might be, at least in part, promoting production of vitamin C via regulating PbrNCED1 expression.

Transcriptomic and evolutionary analyses of white pear (Pyrus bretschneideri) ß-amylase genes reveals their importance for cold and drought stress responses.

ß-amylase (BAM) genes play essential roles in plant abiotic stress responses. Although the genome of Chinese white pear (Pyrus bretschneideri) has recently been made available, knowledge regarding the BAM family in pear, including gene function, evolutionary history and patterns of gene expression remains limited. In this study, we identified 17 PbBAMs in the pear genome. Of these, 12 PbBAM members were mapped onto 9 chromosomes and 5 PbBAM genes were located on scaffold contigs. Based on gene structure, protein motif analysis, and the topology of the phylogenetic tree of the PbBAM family, we classified member genes into 4 groups. All PbBAM genes were found to contain typical glycosyl hydrolysis 14 domain motifs. Interfamilial comparisons revealed that the phylogenetic relationships of BAM genes in other Rosaceae species were similar those found in pear. We also found that whole-genome duplication (WGD)/segmental duplication events played critical roles in the expansion of the BAM family. Next, we used transcriptomic data to study gene expression during the response of drought and low temperate responses, and found that genes in Group B were related to drought and cold stress. We identified four PbBAM genes associated with abiotic stress in Pear. Finally, by analyzing co-expression networks and co-regulatory genes, we found that PbBAM1a and PbBAM1b were associated with the pear abiotic stress response.

A novel MYB transcription factor regulates ascorbic acid synthesis and affects cold tolerance.

Dehydroascorbate reductase (DHAR) plays an important role in stress responses, but the transcriptional regulation of DHAR in response to abiotic stress is still poorly understood. In this study, we isolated a novel R2R3-type MYB transcription factor from Pyrus betulaefolia by yeast one-hybrid screening, designated as PbrMYB5. PbrMYB5 was localized in the nucleus and could bind specifically to the promoter of PbrDHAR2. PbrMYB5 was greatly induced by cold and salt but slightly by dehydration. Overexpression of PbrMYB5 in tobacco conferred enhanced tolerance to chilling stresses, whereas down-regulation of PbrMYB5 in P. betulaefolia by virus-induced gene silencing resulted in elevated chilling sensitivity. Transgenic tobacco exhibited higher expression levels of NtDHAR2 and accumulated larger amount of ascorbic acid (AsA) than the wild-type plants. Virus-induced gene silencing of PbrMYB5 in P. betulaefolia down-regulated PbrDHAR2 abundance and decreased AsA level, accompanied by an increased sensitivity to the chilling stress. Taken together, these results demonstrated that PbrMYB5 was an activator of AsA biosynthesis and may play a positive role in chilling tolerance, at least in part, due to the modulation of AsA synthesis by regulating the PbrDHAR2 expression.