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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 Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02290 • Publication Date (Web): 01 Oct 2017 Downloaded from http://pubs.acs.org on October 4, 2017
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Nanobubble water's promotion effect of barley (Hordeum vulgare L.) sprouts supported by RNA-Seq analysis
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Shu Liu †‡, Seiichi Oshita ‡*, Saneyuki Kawabata ‡*, Dang Quoc Thuyet‡
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†
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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* Corresponding author:
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Seiichi Oshita, E-mail:
[email protected];
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Saneyuki Kawabata, E-mail:
[email protected] 15
TEL: 03-5841-5362
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FAX: 03-5841-8174
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ABSTRACT:
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The physiological promotion effect of nanobubble (NB) water on living organism is a
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still poorly understood phenomenon which was discovered one decade ago. Here, we
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analysed the barley (Hordeum vulgare L.) embryo transcriptome following exposure to NB
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water and low concentration hydrogen peroxide (H2O2) using RNA-Seq. We found that
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349 genes were differentially expressed in response to 24-h exposure of NB water, and 97
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genes were differentially expressed after exposure to H2O2. GO enrichment and cluster
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analyses revealed that NB water-induced expression of genes related to cell division and
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cell wall loosening. RNA-Seq, quantitative real-time polymerase chain reaction (qPCR),
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and enzyme activity measurements all pointed to genes encoding peroxidases as a major
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factor responsible for the effects of physiological enhancement due to NB water. The
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exogenous hydroxyl radical (•OH) produced by NB water significantly increased
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expression of genes related to peroxidase and NADPH, thus leading to an increased
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endogenous superoxide anion (O2·-) inside the barley seed. Appropriately low
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concentrations of exogenously added reactive oxygen species (ROS) and endogenous ROS
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played important roles in plant growth and development. When ROS levels were low, the
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endogenous ROS was eliminated by ascorbate peroxidase (APX) and other peroxidases
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instead of activating the catalase (CAT) and superoxidase dismutase (SOD). This dataset
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will serve as the foundation for a system biology approach to understand NB water’s
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physiological promoting effects on living organisms.
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KEYWORDS: RNA-Seq; Barley seed; Cell wall loosening; Peroxidases; Cell division;
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Hydroxyl radical
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INTRODUCTION
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Bulk Micro- and nanobubbles (MNBs) are gas-filled bubbles that have a diameter of
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micro and nanometer levels 1, 2. In recent years, the research on MNBs has been expanding
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rapidly in various fields due to their unique properties such as long-time stability, a
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significant increase in surface area of high interfacial tension, either negative or positive
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zeta potential and free radical generation. One of the most astonishing effects of bulk MNB
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water is its promotion effect on the physiological activity of living organisms. Although
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numerous experimental and field studies have emerged and successfully confirmed the
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MNB water’s promotion effects on growth of various living organisms, the mechanism for
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this phenomenon remains poorly understood 3-5.
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In our previous study, we reported that without any stimuli, the water containing
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oxygen nanobubbles (NBs) could continually produce a very small amount of reactive
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oxygen species (ROS) in water. The exogenously applied ROS by NB water stimulated the
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generation of endogenous superoxide radical (O2·-) inside the seed and played an important
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role in seed germination 6. By our further research, it has been found that the ROS
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produced by oxygen NB was hydroxyl radical (•OH). The submicromolar level hydroxyl
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radicals produced by NB water have a multitude of effects on vegetable seed germination,
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with the ultimate effect depending on the bubble number density and the seed type
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involved 7.
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In the fields of both plant biology and health and medicine, the newly found role of
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ROS as signalling molecules has been highlighted by accumulating evidence. High
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concentrations of ROS are toxic, but low concentrations of ROS can promote cell
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proliferation and survival
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to very low concentrations of many oxidants, such as superoxide radical (O2·-) and
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hydrogen peroxide (H2O2), stimulated cell growth and division 8. This growth-stimulatory
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effect at low concentration has been seen with human cells 8, 10, mouse cells 11, bacteria
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and yeast 13. In the plant field, it also has been accepted that exogenously supplied H2O2
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can promote germination, as has been shown in barley, wheat, rice and Zinnia elegans
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seeds 14-17.
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. It has been reported that the exposure of the cells in culture
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The connection between signal roles of ROS and the production of ROS by MNB
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water was first proposed by our team to explain NBs’ physiological promotion effects on
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living organisms 6, 7. The mechanism of NBs’ promotion effect on physiological activity of
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living organisms is of great value concerning the clarification to its application and
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prospect. Thus, a deeper molecular biological and genomic analysis is necessary to provide
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a comprehensive understanding of the plant’s response to NB water.
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The advent of next-generation deep-sequencing of RNA molecules (RNA-Seq) has
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made genome-wide transcript analyses both sensitive and quantitative 18. In this study, we
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used RNA sequencing as a tool to obtain detailed expression profiles of genes involved in
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response of the barley seed embryo to NB water. H2O2 solution is also used as a positive
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control. NB water led to the differential expression of 349 transcripts after embryos were
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submerged for 24 h. These included genes involved in cell division, cell proliferation and
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cell wall loosening. We show that NB water has a similar effect on gene expression as
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H2O2 solution. For the entire germination process, the expression of peroxidase genes was
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induced both by NB water and H2O2 solution. Establishing a molecular link between NB
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water and plant growth at the transcriptional level is not only valuable for fundamental
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research, but it also has potential applications.
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EXPERIMENTAL SECTION
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Preparation of NB water
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A MNB generator (OM4-GP-040, Aura Tec Co. Ltd., Japan) was used for producing
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MNBs as described in our previous paper 2. The dissolved oxygen (DO) concentration of
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distilled water was about 8 ppm. To adjust the DO concentration of MNB water to be the
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same as distilled water, the gas-mixture of nitrogen and air was used. During the 60 minutes
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MNB generation, the water has milky white color, as shown in Fig. 1a. Ten minutes after
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the stop of the MNB generation, the water became transparent, as shown in Fig. 1b,
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indicating that almost all of the microbubbles (MBs) disappeared and only NBs remained.
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The germination tests were initiated after approximately 10 minutes after completing MNB
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generation. As MB only can stay in water for a few minutes, we regarded our water as ‘NB
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water’. The total density of nanobubbles in the NB water was above 108 particles/ml, as
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determined by Nano Sight-LM10, Quantum Design Inc., Japan (Fig. 1c). In an MNB
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generator, hydrodynamic cavitation may occur, which can generate H2O2, HNO2, HNO3
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and O3 at a concentration range of 0.001–0.1 mM
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stopped, the cavitation also stopped. Thus, H2O2, HNO2, HNO3 and O3 would not be
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generated after the MNB generator stopped. In our previous study, we have reported that
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the concentration of H2O2 produced through MNB generation was
