Transcriptional and epigenetic analysis reveals that NAC transcription factors regulate fruit flavor ester biosynthesis.

Flavor-associated volatile chemicals make major contributions to consumers' perception of fruits. Although great progress has been made in establishing the metabolic pathways associated with volatile synthesis, much less is known about the regulation of those pathways. Knowledge of how those pathways are regulated would greatly facilitate efforts to improve flavor. Volatile esters are major contributors to fruity flavor notes in many species, providing a good model to investigate the regulation of volatile synthesis pathways. Here we initiated a study of peach (Prunus persica L. Batsch) fruits, and identified that the alcohol acyltransferase PpAAT1 contributes to ester formation. We next identified the transcription factor (TF) PpNAC1 as an activator of PpAAT1 expression and ester production. These conclusions were based on in vivo and in vitro experiments and validated by correlation in a panel of 30 different peach cultivars. Based on homology between PpNAC1 and the tomato (Solanum lycopersicum) TF NONRIPENING (NOR), we identified a parallel regulatory pathway in tomato. Overexpression of PpNAC1 enhances ripening in a nor mutant and restores synthesis of volatile esters in tomato fruits. Furthermore, in the NOR-deficient mutant tomatoes generated by CRISPR/Cas9, lower transcript levels of SlAAT1 were detected. The apple (Malus domestica) homolog MdNAC5 also stimulates MdAAT1 expression via binding to this gene's promoter. In addition to transcriptional control, epigenetic analysis showed that increased expression of NACs and AATs is associated with removal of the repressive mark H3K27me3 during fruit ripening. Our results support a conserved molecular mechanism in which NAC TFs activate ripening-related AAT expression, which in turn catalyzes volatile ester formation in multiple fruit species.

Genome-Wide Analysis of Calmodulin Binding Transcription Activator (CAMTA) Gene Family in Peach (Prunus persica L. Batsch) and Ectopic Expression of PpCAMTA1 in Arabidopsis camta2,3 Mutant Restore Plant Development.

Calmodulin-binding transcription activator (CAMTA) is a transcription factor family containing calmodulin (CaM) binding sites and is involved in plant development. Although CAMTAs in Arabidopsis have been extensively investigated, the functions of CAMTAs remain largely unclear in peaches. In this study, we identified five peach CAMTAs which contained conserved CG-1 box, ANK repeats, CaM binding domain (CaMBD) and IQ motifs. Overexpression in tobacco showed that PpCAMTA1/2/3 were located in the nucleus, while PpCAMTA4 and PpCAMTA5 were located in the plasma membrane. Increased expression levels were observed for PpCAMTA1 and PpCAMTA3 during peach fruit ripening. Expression of PpCAMTA1 was induced by cold treatment and was inhibited by ultraviolet B irradiation (UV-B). Driven by AtCAMTA3 promoter, PpCAMTA1/2/3 were overexpressed in Arabidopsis mutant. Here, we characterized peach PpCAMTA1, representing an ortholog of AtCAMTA3. PpCAMTA1 expression in Arabidopsis complements the developmental deficiencies of the camta2,3 mutant, and restored the plant size to the wild type level. Moreover, overexpressing PpCAMTA1 in camta2,3 mutant inhibited salicylic acid (SA) biosynthesis and expression of SA-related genes, resulting in a susceptibility phenotype to Pst DC3000. Taken together, our results provide new insights for CAMTAs in peach fruit and indicate that PpCAMTA1 is associated with response to stresses during development.

Characterization and Purification of Bergamottin from Citrus grandis (L.) Osbeck cv. Yongjiazaoxiangyou and Its Antiproliferative Activity and Effect on Glucose Consumption in HepG2 cells.

Bergamottin is a natural furanocoumarin compound with weak polarity. Characterization and quantification of bergamottin were carried out in different fruit tissues of various citrus cultivars. Among the four citrus tissues tested, i.e., flavedo, albedo, segment membrane (SM), and juice sacs (JS) in eight citrus cultivars, the highest bergamottin content was found in the flavedo of Citrus grandis (L.) Osbeck cv. Yongjiazaoxiangyou (YJZXY, 666.54 µg·g-1 DW). A combination of silica gel column chromatography and high-speed counter-current chromatography (HSCCC) was established to efficiently purify bergamottin from the flavedo of YJZXY. Bergamottin showed significant antiproliferative activity on three cancer cell lines, i.e., human liver cancer HepG2, promyelocytic leukemia HL-60, and gastric cancer BGC-823 cells, which showed a marked inhibition effect on these cell lines in a dose-dependent manner. In addition, bergamottin significantly increased glucose consumption in HepG2 cells also in a dose-dependent manner, which is the first report of its potential in anti-diabetes applications.

Chilling-induced peach flavor loss is associated with expression and DNA methylation of functional genes.

INTRODUCTION: Flavor is a major contributor to consumer preference. Despite being effective at extending the fruit's commercial life, cold storage also results in a significant loss of flavor volatiles. To date, there has been few studies on the metabolic dynamics and the mechanism underlying the regulatory networks that modulate flavor loss during cold storage for fruit. METHODS: The volatile contents were detected by Gas Chromatography-Mass Spectrometer (GC-MS). Weighted gene co-expression network analysis (WGCNA) was used to identify structure genes and transcription factors (TFs). DNA methylation was analyzed by whole-genome methylation sequencing during cold storage. RESULTS: We generated a temporal map, over hourly to weekly timescales, for the effects of chilling on flavor volatiles by combining metabolome, transcriptome, and DNA methylome in peach fruit. Based on the big data analysis, we developed a regulatory network for volatile formation and found that a decrease in volatiles during cold storage was significantly correlated with a decrease in the expression of synthesis genes. Moreover, TFs associated with these structure genes were identified. Expression of genes involved in ethylene biosynthesis was reduced while cold tolerance pathway was activated in response to low temperature. Functions of those genes were confirmed through transgenic experiments and across peach cultivars, suggesting our dataset is a useful tool for elucidating regulatory factors that have not yet been clarified in relation to flavor and cold tolerance. Genome wide DNA methylation was induced by chilling and peaked at 7 d followed by a decline during 28 d cold storage. Reduction of gene expression was accompanied by major changes in the methylation status of their promoters, including PpACS1, PpAAT1, PpTPS3 and PpMADS2. CONCLUSION: Our study revealed the mechanism for chilling-induced flavor loss of peach fruit through time-course transcriptome and DNA methylome analysis.

Flame Retardant Properties of a Guanidine Phosphate-Zinc Borate Composite Flame Retardant on Wood.

This work combines guanidine dihydrogen phosphate (GDP) and zinc borate (ZB) to modify wood via microwave-ultrasonic impregnation for realizing favorable flame retardancy and thermal stability, which were investigated by the limiting oxygen index (LOI), thermogravimetric analysis (TGA), and cone calorimetry tests (CONE). The treated samples show better performance in fire retardancy with the LOI value increasing to 47.8%, and the results of TGA indicate the outstanding thermal stability of wood. In addition, the decline of heat release rate, total heat release, smoke production rate, and total smoke production examined by CONE further demonstrates the achievement of excellent flame retardancy and smoke suppression properties of the GDP/ZB-modified wood.

Regulation of fruit ripening by the brassinosteroid biosynthetic gene SlCYP90B3 via an ethylene-dependent pathway in tomato.

The essential role of ethylene in fruit ripening has been thoroughly studied. However, the involvement of brassinosteroids (BRs) in the regulation of fruit ripening and their relationship with the ethylene pathway are poorly understood. In the current study, we found that BRs were actively synthesized during tomato fruit ripening. We then generated transgenic lines overexpressing or silencing SlCYP90B3, which encodes a cytochrome P450 monooxygenase that catalyzes the rate-limiting step of BR synthesis. The expression level of SlCYP90B3 was positively related to the contents of bioactive BRs as well as the ripening process in tomato fruit, including enhanced softening and increased soluble sugar and flavor volatile contents. Both carotenoid accumulation and ethylene production were strongly correlated with the expression level of SlCYP90B3, corroborated by the altered expression of carotenoid biosynthetic genes as well as ethylene pathway genes in transgenic tomato fruits. However, the application of the ethylene perception inhibitor 1-methycyclopropene (1-MCP) abolished the promotion effect of SlCYP90B3 overexpression on carotenoid accumulation. Taken together, these results increase our understanding of the involvement of SlCYP90B3 in bioactive BR biosynthesis as well as fruit ripening in tomato, thus making SlCYP90B3 a target gene for improvement of visual, nutritional and flavor qualities of tomato fruits with no yield penalty.

Genome-Wide Identification and Functional Analysis of Carboxylesterase and Methylesterase Gene Families in Peach (Prunus persica L. Batsch).

Carboxylesterases (CXE) and methylesterases (MES) are hydrolytic enzymes that act on carboxylic esters and are involved in plant metabolic processes and defense responses. A few functions of plant CXE and MES genes have been identified but very little information is available about the role of most members. We made a comprehensive study of this gene family in a commercially important species, peach (Prunus persica L. Batsch). A total of 33 peach CXE genes and 18 MES genes were identified and shown to be distributed unevenly between the chromosomes. Based on phylogenetic analysis, CXEs and MESs clustered into two different branches. Comparison of the positions of intron and differences in motifs revealed the evolutionary relationships between CXE and MES genes. RNA-seq revealed differential expression patterns of CXE/MESs in peach flower, leaf, and ripening fruit and in response to methyl jasmonate (MeJA) and ultraviolet B treatment. Transcript levels of candidate genes were verified by real-time quantitative PCR. Heterologous expression in Escherichia coli identified three CXEs that were involved in the hydrolysis of volatile esters in vitro. Furthermore, two recombinant MES proteins were identified that could hydrolyze MeJA and methyl salicylate. Our results provide an important resource for the identification of functional CXE and MES genes involved in the catabolism of volatile esters, responses to biotic and abiotic stresses and activation of signaling molecules such as MeJA and methyl salicylate.

Peach Carboxylesterase PpCXE1 Is Associated with Catabolism of Volatile Esters.

Peach fruit volatile acetate esters impact consumer sensory preference and contribute to defense against biotic stresses. Previous studies showed that alcohol acyltransferase (AAT) family PpAAT1 is correlated with volatile ester formation in peach fruits. However, fruits also contain carboxylesterase (CXE) enzymes that hydrolyze esters. The functions of this family with regard to volatile ester content has not been explored. Here, we observed that content of acetate ester was negatively correlated with expression of PpCXE1. Recombinant PpCXE1 protein exhibited hydrolytic activity toward acetate esters present in peach fruit. Kinetic analysis showed that PpCXE1 showed the highest catalytic activity toward E-2-hexenyl acetate. Subcellular localization demonstrated that PpCXE1 is present in the cytoplasm. Transient expression in peach fruit and stable overexpression in tomato fruit resulted in significant reduction of volatile esters in vivo. Taken together, the results indicate that PpCXE1 expression is associated with catabolism of volatile acetate esters in peach fruit.

Neuroprotective effects of lotus seedpod procyanidins on extremely low frequency electromagnetic field-induced neurotoxicity in primary cultured hippocampal neurons.

The present study investigated the protective effects of lotus seedpod procyanidins (LSPCs) on extremely low frequency electromagnetic field (ELF-EMF)-induced neurotoxicity in primary cultured rat hippocampal neurons and the underlying molecular mechanism. The results of MTT, morphological observation, superoxide dismutase (SOD) and malondialdehyde (MDA) assays showed that compared with control, incubating neurons under ELF-EMF exposure significantly decreased cell viability and increased the number of apoptotic cells, whereas LSPCs evidently protected the hippocampal neurons against ELF-EMF-induced cell damage. Moreover, a certain concentration of LSPCs inhibited the elevation of intracellular reactive oxygen species (ROS) and Ca(2+) level, as well as prevented the disruption of mitochondrial membrane potential induced by ELF-EMF exposure. In addition, supplementation with LSPCs could alleviate DNA damage, block cell cycle arrest at S phase, and inhibit apoptosis and necrosis of hippocampal neurons under ELF-EMF exposure. Further study demonstrated that LSPCs up-regulated the activations of Bcl-2, Bcl-xl proteins and suppressed the expressions of Bad, Bax proteins caused by ELF-EMF exposure. In conclusion, these findings revealed that LSPCs protected against ELF-EMF-induced neurotoxicity through inhibiting oxidative stress and mitochondrial apoptotic pathway.

Chemoprotective action of lotus seedpod procyanidins on oxidative stress in mice induced by extremely low-frequency electromagnetic field exposure.

With the increasing use of electromagnetic technology, the effects of extremely low-frequency electromagnetic fields (ELF-EMF) on biological systems, central neurotransmitter systems, and human health have attracted extensive attention worldwide. In this study, lotus seedpod procyanidins (LSPCs) were evaluated for their protective effects on ELF-EMF induced oxidative stress injury in mice. Sixty male ICR mice were used for the experiment. The mice were randomly divided into five equal groups. The control group did not receive LSPCs or ELF-EMF but orally received normal saline. The ELF-EMF group received ELF-EMF exposure plus normal saline orally. The other three groups received ELF-EMF exposure plus LSPCs orally (60, 90, or 120mg kg(-1).bw, respectively). Each group exposed to ELF-EMF at 8 mT, 4h day(-1) for 28 consecutive days after administration daily of LSPCs or normal saline to mice for 15 consecutive days with the exception of the control group. Thereafter, blood and cerebral cortex of the mice were analyzed for antioxidant indices, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione-S-transferase (GST) and malondialdehyde (MDA). LSPCs administration at different doses significantly inhibited oxidative stress damage of mice induced by ELF-EMF. LSPCs treatment augmented SOD, CAT, GSH-Px, GR and GST activity. Furthermore, administration significantly lowered MDA level in LSPCs treatment groups LSPCs. All results indicated LSPCs can effectively prevent oxidative stress injury induced by ELF-EMF exposure, which may be related to its ability of scavenging free radicals and stimulating antioxidant enzyme activity.