Comparative Transcriptome Analysis of Softening and Ripening-Related Genes in Kiwifruit Cultivars Treated with Ethylene.

This work presents the transcriptome analysis of green 'Hayward' (Actinidia deliciosa) and gold 'Haegeum' (Actinidia chinensis) kiwifruit cultivars after treatment with ethylene for three days at 25 °C. Illumina high-throughput sequencing platform was used to sequence total mRNAs and the transcriptome gene set was constructed by de novo assembly. A total of 1287 and 1724 unigenes were differentially expressed during the comparison of ethylene treatment with control in green 'Hayward' and gold 'Haegeum', respectively. From the differentially expressed unigenes, 594 and 906 were upregulated, and 693 and 818 were downregulated in the green and gold kiwifruit cultivars, respectively, when treated with ethylene. We also identified a list of genes that were expressed commonly and exclusively in the green and gold kiwifruit cultivars treated with ethylene. Several genes were expressed differentially during the ripening of kiwifruits, and their cumulative effect brought about the softening- and ripening-related changes. This work also identified and categorized genes related to softening and other changes during ripening. Furthermore, the transcript levels of 12 selected representative genes from the differentially expressed genes (DEGs) identified in the transcriptome analysis were confirmed via quantitative real-time PCR (qRT-PCR) to validate the reliability of the expression profiles obtained from RNA-Seq. The data obtained from the present study will add to the information available on the molecular mechanisms of the effects of ethylene during the ripening of kiwifruits. This study will also provide resources for further studies of the genes related to ripening, helping kiwifruit breeders and postharvest technologists to improve ripening quality.

Transcriptome Analysis of Pre-Storage 1-MCP and High CO2-Treated 'Madoka' Peach Fruit Explains the Reduction in Chilling Injury and Improvement of Storage Period by Delaying Ripening.

Cold storage of peach fruit at low temperatures may induce chilling injury (CI). Pre-storage 1-MCP and high CO2 treatments were reported among the methods to ameliorate CI and reduce softening of peach fruit. However, molecular data indicating the changes associated with pre-storage 1-MCP and high CO2 treatments during cold storage of peach fruit are insufficient. In this study, a comparative analysis of the difference in gene expression and physico-chemical properties of fruit at commercial harvest vs. stored fruit for 12 days at 0 °C (cold-stored (CS), pre-storage 1-MCP+CS, and pre-storage high CO2+CS) were used to evaluate the variation among treatments. Several genes were differentially expressed in 1-MCP+CS- and CO2+CS-treated fruits as compared to CS. Moreover, the physico-chemical and sensory data indicated that 1-MCP+CS and CO2+CS suppressed CI and delayed ripening than the CS, which could lead to a longer storage period. We also identified the list of genes that were expressed commonly and exclusively in the fruit treated by 1-MCP+CS and CO2+CS and compared them to the fruit quality parameters. An attempt was also made to identify and categorize genes related to softening, physiological changes, and other ripening-related changes. Furthermore, the transcript levels of 12 selected representative genes from the differentially expressed genes (DEGs) in the transcriptome analysis were confirmed via quantitative real-time PCR (qRT-PCR). These results add information on the molecular mechanisms of the pre-storage treatments during cold storage of peach fruit. Understanding the genetic response of susceptible cultivars such as 'Madoka' to CI-reducing pre-storage treatments would help breeders release CI-resistant cultivars and could help postharvest technologists to develop more CI-reducing technologies.

Ascorbic Acid Induces Necrosis in Human Laryngeal Squamous Cell Carcinoma via ROS, PKC, and Calcium Signaling.

Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/ß activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 232: 417-425, 2017. © 2016 Wiley Periodicals, Inc.

Preharvest Methyl Jasmonate Treatment Affects the Mineral Profile, Metabolites, and Antioxidant Capacity of Radish Microgreens Produced without Substrate.

This study investigated the impact of Methyl Jasmonate (MeJA) application on the nutritional content and yield of five different colored radish microgreens. Microgreens were produced without substrate and subjected to 0.5 mM and 1.0 mM MeJA treatments on the 7th day, three days before harvest. The parameters measured included yield, dry matter, minerals, amino acids, secondary metabolites such as chlorophylls (Chls), anthocyanins, flavonoids, phenolics, glucosinolates (GSLs), vitamin C, and antioxidant capacity. MeJA at 1.0 mM generally improved yield and dry weight across cultivars, and all microgreens exhibited rich mineral and amino acid composition, with the influence of cultivar being more significant than MeJA treatment. However, MeJA enhanced all cultivars' anthocyanins, GSLs, phenolics, flavonoids, and antioxidant activities. Generally, as the antioxidant capacity is the primary factor influencing the nutritional quality of microgreens, MeJA-treated microgreens, especially with selected superior cultivars such as 'Asia purple' and 'Koregon red', could offer a potential for cultivation of value-added, eco-friendly microgreens with substrate-free cultivation.

CoCl2 induces apoptosis through the mitochondria- and death receptor-mediated pathway in the mouse embryonic stem cells.

Embryonic hypoxia/ischemia is a major cause of a poor fetal outcome and future neonatal and adult handicaps. However, biochemical cellular events in mouse embryonic stem (mES) cells during hypoxia remains unclear. This study investigated the underlying mechanism of apoptosis in mES cells under CoCl2-induced hypoxic/ischemic conditions. CoCl2 enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and the accumulation of reactive oxygen species in mES cells. The CoCl2-treated mES cells showed a decrease in cell viability as well as typical apoptotic changes, cell shrinkage, chromatin condensation, and nuclear fragmentation and an extended G2/M phase of the cell cycle. CoCl2 augmented the release of cytochrome c into the cytosol from the mitochondria with a concomitant loss of the mitochondrial transmembrane potential (ΔΨm) and upregulated the voltage-dependent anion channel. In addition, CoCl2-induced caspase-3, -8, and -9 activation and upregulation of p53 level, whereas downregulated Bcl-2 and Bcl-xL, a member of the anti-apoptotic Bcl-2 family in mES cells. Furthermore, CoCl2 led to the upregulation of Fas and Fas-ligand, which are the death receptor assemblies, as well as the cleavage of Bid in mES cells. These results suggest that CoCl2 induces apoptosis through both mitochondria- and death receptor-mediated pathways that are regulated by the Bcl-2 family in mES cells.

Prediction of tannin content and quality parameters in astringent persimmons from visible and near-infrared spectroscopy.

Introduction: Tannin content and postharvest quality characteristics of persimmon fruit are often determined by the destructive analysis that consumes time, does not allow the acquisition of data from the same fruit continuously, and requires expensive high-performance equipment. This research was done to investigate the potential for non-destructive estimation of astringency and quality parameters in persimmon fruit based on visible/near-infrared (VNIR) spectra. Methods: VNIR spectra readings, the reference tannin content, and quality parameters were measured from fruits of "Cheongdo-Bansi" and "Daebong" persimmon cultivars at harvest and throughout the ripening/deastringency period. The spectra readings from half of the total fruit were utilized for the calibration set, while the other half readings were used for the prediction set. To develop models correlating the spectra data to the measured reference parameters data, the partial least square regression (PLSR) method was utilized. Results and discussion: In the case of 'Daebong', the coefficients of determination (R2) between VNIR spectra and the actual measured values of TSS, firmness, simple sugars, and tannin content were (0.95, 0.94, 0.96, and 0.96) and (0.93, 0.89, 0.96, and 0.93), for the calibration and prediction sets, respectively. Similarly, the R2-values of (0.86, 0.93, 0.79, and 0.81) and (0.83, 0.91, 0.75, and 0.75) were recorded in 'Cheongdo-Bansi' for the calibration and prediction sets, respectively. Additionally, the acquired data were divided into two sets in a 3:1 ratio to develop predictive models and to validate the models in multiple regressions. PLSR models were developed in multiple regression to estimate the tannin content of both cultivars from firmness and simple sugars with R2-values of 0.83 and 0.79 in 'Cheongdo-Bansi' for the calibration and prediction sets, respectively, whereas, R2-values of 0.80 and 0.84 were recorded in 'Daebong' for the calibration and prediction sets, respectively. The overall findings of this study showed the possibility of using VNIR spectra for the prediction of postharvest quality and tannin contents from intact persimmon fruit with quick, chemical-free, and low-cost assessment methods. Also, the multiple regression using physicochemical parameters could fairly predict the tannin content in persimmon fruit though destructively but save time and low-cost.

Radish microgreens produced without substrate in a vertical multi-layered growing unit are rich in nutritional metabolites.

Growing microgreens on trays without substrate in a vertical multilayered growing unit offers several advantages over traditional agriculture methods. This study investigated the yield performance and nutritional quality of five selections of radish microgreens grown in sprouting trays, without a substrate using only water, in an indoor multilayer cultivation system using artificial light. Various parameters were measured, including fresh weight, dry matter, chlorophyll, minerals, amino acids, phenolics, flavonoids, anthocyanins, vitamin C, glucosinolates, and antioxidant activity with four different in vitro assays. After ten days, the biomass had increased by 6-10 times, and the dry matter varied from 4.75-7.65%. The highest yield was obtained from 'Asia red', while the lowest was from 'Koregon red'. However, 'Koregon red' and 'Asia red' had the highest dry matter. 'Asia red' was found to have the highest levels of both Chls and vitamin C compared to the other cultivars, while 'Koregon red' exhibited the highest levels of total phenolics and flavonoids. Although variations in the levels of individual glucosinolates were observed, there were no significant differences in the total content of glucosinolates among the five cultivars. 'Asia purple' had the highest anthocyanin content, while 'Asia green 2' had the lowest. The K, Mg, and Na concentrations were significantly highest in 'Asia green 2', and the highest Ca was recorded in 'Asia purple'. Overall, 'Asia purple' and 'Koregon red' were the best cultivars in terms of nutritional quality among the tested radish microgreens. These cultivars exhibited high levels of dry weight, total phenolics, flavonoids, anthocyanins, essential and total amino acids, and antioxidant activities. Moreover, the implementation of this vertical cultivation method for microgreens, which relies solely on water and seeds known for their tall shoots during the sprouting could hold promise as a sustainable approach. This method can effectively be utilized for cultivar screening and fulfilling the nutritional and functional needs of the population while minimizing the environmental impacts associated with traditional agriculture practices.

Changes in metabolites and antioxidant activities of green 'Hayward' and gold 'Haegeum' kiwifruits during ripening with ethylene treatment.

Fruit ripening involves changes in physical, physiological, biochemical, and metabolic activities through the actions of enzymes and regulatory genes. In this study, we elucidate primary and secondary metabolites and antioxidant activities of green 'Hayward' and gold 'Haegeum' kiwifruit cultivars during ripening by comparing ethylene-treated fruit to the control. A total of 36 primary metabolites (20 amino acids, 9 fatty acids, 4 organic acids, and 3 sugars) were identified to be altered significantly during the ripening of both cultivars. Significant changes in secondary metabolites such as total phenols, flavonoids, and vitamin C were also observed during the ripening of both cultivars. Moreover, antioxidant activity assays showed that ripening either maintains or increases the antioxidant activity of kiwifruit cultivars. These findings could assist the fruit industry in developing metabolic markers for indication of the optimum kiwifruit ripening quality and postharvest decisions for storage, distribution, and marketing.

Prestorage High CO2 and 1-MCP Treatment Reduce Chilling Injury, Prolong Storability, and Maintain Sensory Qualities and Antioxidant Activities of "Madoka" Peach Fruit.

Cold storage is widely used to prolong the storability of peach fruit. However, prolonged storage at low temperatures results in chilling injury (CI) in some susceptible peach cultivars during or after cold storage. Prestorage high CO2 and 1-methylcyclopropene (1-MCP) treatments are among the methods reported to alleviate CI and maintain the firmness of peach fruit. Hence, this study investigated CI, ripening-related physicochemical parameters, sensory qualities, total phenolics and flavonoids, and antioxidant activities of "Madoka" peach fruit to observe the effectiveness of prestorage treatment with high CO2 and 1-MCP during the storage at 0 and 5°C. Based on the CI index, control fruits were acceptable for marketing up to 20 and 16 days of storage at 0 and 5°C, respectively, while the treated fruits could be marketable up to 28 days of storage. The results of firmness and firmness-related parameters [pectin content and polygalacturonase (PG) activity] also revealed that both high CO2 and 1-MCP treatments were effective in delaying the ripening process of Madoka peach, and the storage at 0°C showed better results than at 5°C. However, based on the overall sensory evaluation results, the treated and control fruits were acceptable for marketing up to 20 and 12 days of storage, respectively, in both storage conditions. After deciding on fruit marketability based on the combined objective postharvest quality parameters and subjective sensory qualities, we analyzed the changes in total phenolics, flavonoids, and antioxidant activities at harvest, on the 12 and 20th days of cold storage. Storage of Madoka peach at 0°C maintained total phenolics, flavonoids, and antioxidant activities regardless of prestorage treatment with high CO2 and 1-MCP. In summary, storing Madoka peach fruit at 0°C after treating it with 30% CO2 for 6 h or 0.5 µl L-1 1-MCP for 24 h reduces CI, prolongs storability, and maintains sensory quality and antioxidant properties.

Preharvest Methyl Jasmonate Treatment Increased the Antioxidant Activity and Glucosinolate Contents of Hydroponically Grown Pak Choi.

Vertical hydroponics farming has emerged as an alternative solution to feed the continuously growing world population. Additionally, recent studies reported that the exogenous treatments of jasmonic acid influence the phytochemical composition of Brassicaceae. We conducted this study to determine the effect of preharvest methyl jasmonate (MeJA) treatment on the phytochemical composition and antioxidant activities of soil- and hydroponically grown pak choi. An aqueous solution of 0.5-mM MeJA was sprayed to saturation on the aerial plant part three days before harvest. The harvested pak choi was freeze-dried and then powdered to measure the antioxidant activity and the contents of chlorophylls (Chls), total phenolics and flavonoids, and glucosinolates (GSLs). The overall results revealed that pak choi grown in vertical hydroponics had higher total Chls and total phenolics than those grown in soil in the greenhouse, regardless of MeJA treatment. Nevertheless, the GSLs content and total flavonoids increased significantly due to MeJA treatment in both growing systems, and the highest values were recorded in hydroponically grown MeJA-treated pak choi. Similarly, the 2, 2-di-phenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, Trolox-equivalent antioxidant capacity (ABTS), oxygen radical absorbance capacity (ORAC), and ferric-reducing antioxidant power (FRAP) were highest in hydroponically grown MeJA-treated pak choi. Taken together, the preharvest foliar treatment of MeJA can be used to improve the phytochemical composition of pak choi grown in both growing systems. Interestingly, the results strongly support the use of MeJA treatment in the vertical hydroponics growing system compared to the conventional growing system in the soil. This indicates that supplementing the vertical hydroponic growing system with preharvest MeJA treatment could be the best option to improve both the yield per square meter and the quality of pak choi. Besides, MeJA-treated pak choi could be used as a value-added horticultural commodity, as its antioxidant activity increased after treatment. Moreover, after further studies, MeJA could also be applied to other Brassica vegetables to improve their GSL contents and antioxidant properties.

Intermolecular cross-talk between NTR1 and NTR2 neurotensin receptor promotes intracellular sequestration and functional inhibition of NTR1 receptors.

G-protein-coupled receptors (GPCR) are now regarded as being able to acquire heterodimer conformations affecting their pharmacology, signaling and trafficking. In co-immunoprecipitation studies using differentially epitope-tagged receptors, we herein provide direct evidence for heterodimerization of human neurotensin type 1 receptor (hNTR1) and type 2 receptor (hNTR2). Using chimeric constructs, we also identified the hNTR2 transmembrane 2 (TM2) to TM4 region as crucial for the formation of the dimerization interface. At the functional level, we demonstrated that the co-expression of hNTR2 suppressed hNTR1-mediated adenylate cyclase/cAMP and phospholipase C activation. Finally, confocal microscopy revealed that whereas tagged hNTR1 expressed alone were localized to the plasma membrane, co-expression of hNTR2 caused the retention of hNTR1 in sub-cellular compartments, indicating that heterodimerization with hNTR2 interferes with the proper recruitment of hNTR1 to the plasma membrane. Overall, this study proposes a novel function of NTR2 in the regulation of NTR1 activity.

B-cell translocation gene 2: expression in the rat ovary and potential association with adenine nucleotide translocase 2 in mitochondria.

The B-cell translocation gene 2 (Btg2) is an anti-proliferative tumor suppressor gene that behaves as a transcriptional regulator. The present study investigated gonadotropin induction of Btg2 in the rat ovary and the mechanism of Btg2 action as a partner of mitochondrial protein adenine nucleotide translocase 2 (Ant2). Transient induction of Btg2 as well as Btg1 mRNA levels by LH/hCG was observed in ovarian granulosa cells. Btg2 protein levels were also stimulated by LH/hCG. LH-induced gene expression of Btg2 required ERK signal pathway. Studies of deletion mutants in HeLa cells showed that deletion of Btg2 C-terminus (Btg2/ΔC) abolished the interaction with Ant2. In fact, the expression levels of Btg2/ΔC construct were decreased in mitochondrial fraction. Btg2 was also expressed in mitochondria and interacted with Ant2 in preovulatory granulosa cells. Interestingly, a Btg2/ΔC construct inhibited an action of Btg2 wild-type on ATP and H(2)O(2) production. These findings demonstrate the gonadotropin stimulation of Btg2 in the ovary and, the physical interaction of Btg2 with Ant2 in mitochondria.