Article Cite This: Langmuir 2017, 33, 12478-12486
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Nanobubble Water’s Promotion Effect of Barley (Hordeum vulgare L.) Sprouts Supported by RNA-Seq Analysis Shu Liu,†,‡ Seiichi Oshita,*,‡ Saneyuki Kawabata,*,‡ and Dang Quoc Thuyet‡ †
Department of Environmental Science and Engineering, School of Space and Environment, Beihang University, Beijing 10191, China Graduate School of Agricultural & Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
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ABSTRACT: The physiological promotion effect of nanobubble (NB) water on living organisms is still a poorly understood phenomenon which was discovered 1 decade ago. Here, we analyzed the barley (Hordeum vulgare L.) embryo transcriptome following the exposure to NB water and lowconcentration hydrogen peroxide (H2O2) using RNA-Seq. We found that 349 genes were differentially expressed after 24 h exposure to NB water and 97 genes were differentially expressed after exposure to H2O2 solution. Gene ontology enrichment and cluster analyses revealed that NB water induced expression of genes related to cell division and cell wall loosening. RNA-Seq, quantitative real-time polymerase chain reaction, and enzyme activity measurements all pointed to gene-encoding peroxidases as a major factor responsible for the effects of physiological enhancement due to NB water. The exogenous hydroxyl radical (•OH) produced by NB water significantly increased the expression of genes related to peroxidase and NADPH, thus leading to an increased endogenous superoxide anion (O2•−) inside the barley seed. Appropriately, low concentrations of exogenously added reactive oxygen species (ROS) and endogenous ROS played important roles in plant growth and development. When ROS levels were low, the endogenous ROS was eliminated by ascorbate peroxidase and other peroxidases instead of activating the catalase and superoxidase dismutase. This data set will serve as the foundation for a system biology approach to understand physiological promotion effects of NB water on living organisms.
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INTRODUCTION
germination, with the ultimate effect depending on the bubble number density and the seed type involved.7 In the fields of both plant biology and health and medicine, the newly found role of ROS as signaling molecules has been highlighted by accumulating evidence. High concentrations of ROS are toxic, but low concentrations of ROS can promote cell proliferation and survival.8,9 It has been reported that the exposure of cells in culture to very low concentrations of many oxidants, such as superoxide radical (O2•−) and hydrogen peroxide (H2O2), stimulated cell growth and division.8 This growth stimulatory effect at low concentrations has been seen with human cells,8,10 mouse cells,11 bacteria,12 and yeast.13 In the plant field, it has also been accepted that exogenously supplied H2O2 can promote germination, as has been shown in barley, wheat, rice, and Zinnia elegans seeds.14−17 The connection between signal roles of ROS and the production of ROS by MNB water was first proposed by our team to explain the physiological promotion effects of NBs on living organisms.6,7 The mechanism of promotion effect of NB on the physiological activity of living organisms is of great value concerning the clarification to its application and prospect.
Bulk micro- and nanobubbles (MNBs) are gas-filled bubbles that have a diameter of micro- and nanometer levels.1,2 In recent years, the research on MNBs has been expanding rapidly in various fields owing to their unique properties such as longtime stability, a significant increase in the surface area of high interfacial tension, either negative or positive zeta potential, and free radical generation. One of the most astonishing effects of bulk MNB water is its promotion effect on the physiological activity of living organisms. Although numerous experimental and field studies have emerged and successfully confirmed the promotion effects of MNB water on the growth of various living organisms, the mechanism for this phenomenon remains poorly understood.3−5 In our previous study, we reported that without any stimuli, the water containing oxygen nanobubbles (NBs) could continually produce a very small amount of reactive oxygen species (ROS) in water. The exogenously applied ROS by NB water stimulated the generation of endogenous superoxide radical (O2•−) inside the seed and played an important role in seed germination.6 By our further research, it has been found that the ROS produced by oxygen NB was a hydroxyl radical (•OH). The submicromolar-level hydroxyl radicals produced by NB water have a multitude of effects on vegetable seed © 2017 American Chemical Society
Received: July 3, 2017 Revised: September 30, 2017 Published: October 1, 2017 12478
DOI: 10.1021/acs.langmuir.7b02290 Langmuir 2017, 33, 12478−12486
Article
Langmuir
Figure 1. Properties of MNB water and NB water. The MNB consists of a mixture of nitrogen and air. (a) Image of MNB water just after the stop of the generation. (b) Image of NB water after approximately 10 min after completing MNB generation, within the period, the MBs disappeared. (c) Bubble size distribution in gas-mixture NBs after more than 10 min of 1 h generation. Seed Germination. Seeds of barley (Hordeum vulgare L.), which were harvested in 2013 and stored under controlled conditions (room temperature), were obtained from the Institute for Sustainable Agroecosystem Services of the University of Tokyo. Germination tests were performed repeatedly with three seed groups. Each group was composed of 50 seeds. We used distilled water as a negative control and 0.3 mM H2O2 solution as a positive control. Barley seeds were submerged in beakers filled with distilled water, NB water, and H2O2 solution in a ratio of 10 mL of water per seed. During the germination experiments, NB water, distilled water, and H2O2 solution were changed twice daily to avoid lack of oxygen and to maintain a certain amount of NBs in water. Germination tests lasted for approximately 2 days. RNA Isolation. Following the submergence of seeds for different times (6, 12, 15, 18, and 24 h), total RNA was extracted from barley embryos, according to the manufacturer’s protocol (Spectrum Plant Total RNA Kit, Sigma). For each replication, 100 mg of barley seed embryos (from 40 seeds) were used. For each treatment, three biological replicates were performed. RNA concentration of each sample was quantified with Qubit assays (Qubit 3.0 Fluorometer, Thermo Fisher Scientific Inc.). RNA-Seq Analysis. Samples from 6 and 24 h treatments were used for RNA-Seq experiments. The same amounts of three replications of each treatment were mixed together and used for deep sequencing and generation of datasets. RNA levels were quantified, and 50 μg of the RNA was sequenced using Hiseq4000 (Illumina, Inc.). The barley genome sequence and the annotation data were downloaded. TopHat was used to align the RNA-Seq reads to the barley reference genome. Then, the clean reads were mapped against the reference genome and statistical analyses. Cufflinks was used to assemble transcripts and estimate their abundance as fragments per kilobase of transcript per million mapped read (FPKM) values. The Cuffdiff program was used to determine differential gene expression analysis.22 Gene Ontology Enrichment and Clustering Analysis. Differentially expressed genes were identified by both p value and foldchange criteria.23 A p value of 0.05 and a relative value of the log 2 ratio ≥2 provided the significance thresholds for gene expression differences in our experiments. Gene ontology (GO) terms were analyzed by g:Profiler (http://biit.cs.ut.ee/gprofiler/). The results were visualized using the R package “REVIGO.”24 Cluster analysis for gene expression profiles at two germination stages (6 and 24 h treatments) and three different treatments (distilled water, NB water, and H2O2 solution) was also performed using R package MBCluster.Seq.25 For the cluster analysis, a false discovery rate (q value)
