Do cucumbers sleep during Witching hour?

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Osamu Takagi, Masamichi Sakamoto, Kimiko Kawano, Mikio Yamamoto This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3736017/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In recent years, several lines of evidence have been reported indicating that brainless organisms also sleep. In this paper, with the aim of answering the question of whether brainless plants also sleep, we analyzed the relationship between the diurnal time at which cucumber sections were prepared and the concentration of gas released from the sections within a certain period of time thereafter. The results showed that the gas concentration released from cucumber sections prepared around 2:00 a.m., the middle of the night, known in Japan as "Ushi-Mitsu-Doki", was the lowest of the day. Our results suggested that the gas production reaction of cucumbers changes depending on the time of sectioning, and that cucumbers were in a state resembling sleep at the "Ushi-Mitsu-Doki" time. Biological sciences/Plant sciences Earth and environmental sciences/Environmental sciences Figures Figure 1 Figure 2 Figure 3 One-Sentence Summary Even cucumbers, which have no brain, sleep in the middle of the night. INTRODUCTION For many years, it was believed that sleep is a system controlled by the brain, and that animals without brains do not sleep. Recently, however, jellyfish and hydras have been shown to sleep, and several lines of evidence have been reported that even brainless organisms can sleep ( 1 – 3 ). There is also a Japanese proverb about the late night time from about 2:00 a.m. to 2:30 a.m. which coincides with the “witching hour”. The proverb states "Kusa-Ki-Mo-Nemuru-Ushi-Mitsu-Doki" which translates to “a time so quiet that not only people and animals but also grass and trees fall asleep”. Vegetables and fruits, which are plants, have been reported to have long-lasting biological responses after harvest ( 4 ) including the release of several types of volatiles when plants are exposed to stimuli or trauma after harvest ( 5 , 6 ). The released volatiles in response to stimuli and trauma are believed to be plant-to-plant communications to elicit resistance against external enemies, to repair, and to promote immunological defenses. ( 7 – 16 ). We analyzed the concentrations of volatiles released from cucumber sections ( Cucumis sativus ) after harvest and reported that the circadian rhythm of gas concentrations varied with different seasons ( 17 – 19 ). In this paper, with the aim of answering the question of whether plants without brains also sleep, we analyzed the relationship between the diurnal time at which cucumber sections were prepared and the concentration of gas released from the sections within a designated period of time thereafter. As a result, we report that the gas concentration released from cucumber sections prepared at the "Ushi-Mitsu-Doki", or the Witching hour, was the lowest of the day. SAMPLE PREPARATION AND GAS CONCENTRATION MEASUREMENT Figures 1 (a) and 1(b) show photographs of four uniform samples A 1 , A 2 , B 1 , and B 2 prepared from four cucumbers. To prepare four uniform samples, we put one section from each of the four cucumbers into each Petri dish, for a total of four sections per dish. Here, A 1 and A 2 contain slices that share one cut surface which were positioned with that surface facing upwards. B 1 and B 2 are also similarly prepared, sharing single cut surfaces. Figure 1 (c) shows the Petri dishes placed in two layers after sample preparation. After leaving the Petri dishes as illustrated in Fig. 1 (c) for 30 minutes, the lids were removed from the Petri dishes, and the dishes containing the samples were placed in a sealed polypropylene container with a volume of 2.2 liters, and stored for 24 to 48 hours in a temperature-controlled (22 to 24 degrees Celsius) room with no direct sunlight. Although volatile components are released from the sample in the sealed container, the gas concentration reaches a maximum in about 12 hours, after which the measured gas concentration is found to remain in equilibrium ( 20 ). While the gas concentration remained at equilibrium, the concentration of hexanol gas was measured by aspirating 300 ml of gas from the sealed container using a gas detector tube (141L: GASTEC, Japan) and a gas detector device (GV-100: GASTEC). MEASUREMENT AND ANALYSIS OF GAS CONCENTRATIONS Figure 2 shows the results of gas concentration measurements. The horizontal axis is the 24-hour period from 12:00 noon to 12:00 noon the next day. Here, the time represents the time when the four samples A 1 , A 2 , B 1 , and B 2 were prepared. The vertical axis is gas concentration (ppm). In Fig. 2 , 468 data points are plotted, each point being the average of the gas concentrations emitted from the four samples A 1 , A 2 , B 1 , and B 2 . Figure 3 shows in red the moving average of the gas concentrations shown in Fig. 2 . We used the weighted moving average with the Gaussian function as weights. Here, σ = 0.5 hours was adopted for the calculation where σ 2 of the Gaussian function was used as the variance. The average of the 468 gas concentration data, 353.8 ppm, is indicated by the blue line. RESULTS AND DISCUSSION Figure 3 shows that the gas concentration emitted from the cucumber section varies depending on the time when the sample was prepared. Characteristically, the gas concentration reached a maximum around 15:00 and a minimum around 2:00 after midnight. This indicates that the gas production reaction is most active when cucumbers are cut around 15:00, gradually suppressed after 15:00, and most suppressed around 2:00 in a 24-hour day. From these results the following 1 to 3 can be inferred. That plant-to-plant communication becomes most active around 15:00. It appears that cucumber plants consider this time of day to be the most dangerous time and that communication and defenses need to be the most active around this time. Cucumbers sense that around the 2:00 a.m. time, the need for inter-plant communication is at its lowest. The increase in the moving average of gas concentrations from 2:00 to 6:00 indicates that the biological response is activated and this correlates with coming sunrise. CONCLUSION We sliced cucumbers and measured the gas concentrations of volatile components emitted from the cucumber sections. Analysis of the relationship between the time of cucumber cutting and gas concentration revealed that the intensity of the gas production reaction varied depending on the diurnal time when the cut was performed. In particular, the response was most active around 15:00 and most suppressed around 2:00. We concluded that cucumbers mimic a sleep-like state around 2:00, because this is the time when the gas production reaction of cucumbers is highly suppressed. In addition, since around two o’clock corresponds to the Japanese word “Ushi-Mitsu-Doki”, we proved that the Japanese proverb "Kusa-Ki-Mo-Nemuru-Ushi-Mitsu-Doki" which means that grass and trees fall asleep at Ushi-Mitsu-Doki is indeed correct. Declarations Acknowledgments: This research was funded by Science Peace Culture Foundation (SPC-F), (Chairman of the Board of Directors: Dr. Mikio Yamamoto). Author Contributions : O.T. designed and performed the experiments. O.T. analyzed data and prepared the samples. O.T. wrote the paper. All authors discussed the experiments, analytical methods, and results and commented on the manuscript. Competing interests: Authors declare that they have no competing interests. Data and materials availability: All data are available in the main text or the supplementary materials. References Ravi, D. N., Claire, N. B., Michael J. A., Ty, B., Justin, S. B., David, A. P., Paul, W. S., Viviana, G., Lea, G. (2017) The Jellyfish Cassiopea Exhibits a Sleep-like State. Current Biology. 27, 2984–2990. https://doi.org/10.1016/j.cub.2017.08.014 John, A., Lesku, L. M. T. L. (2017) Sleep Origins: Restful Jellyfish Are Sleeping Jellyfish. Current Biology. 27, R1060-R1062. https://doi.org/10.1016/j.cub.2017.08.024 Hiroyuki, J. K., Sungeon, P., Ji-Hyung, K., Junko, K., Sofian, K., Etsuko, S., Aya, S., Jongbin, L., Hyunwoo, B., Yoshitaka, K., Chunghun, L., Taichi, Q. I. (2020) A sleep-like state in Hydra unravels conserved sleep mechanisms during the evolutionary development of the central nervous system. Science Advances. 6, eabb9415. https://doi.org/10.1126/sciadv.abb9415 Goodspeed, D., Liu, J.D., Chehab, E.W., Sheng, Z., Francisco, M., Kliebenstein, D.J. & Braam, J. (2013) Postharvest Circadian Entrainment Enhances Crop Pest Resistance and Phytochemical Cycling. Current Biology. 23, 1235–1241. https://doi.org/10.1016/j.cub.2013.05.034 Hatanaka, A. (1993) The biogeneration of green odor by green leaves. Phytochemistry, 34, 1201–1218. https://doi.org/10.1016/0031-9422(91)80003-J Hatanaka, A. (1996) The fresh green odor emitted by plants. Food Reviews International, 12, 303–350. https://doi.org/10.1080/87559129609541083 Goodspeed, D., Liu, J.D., Chehab, E.W., Sheng, Z., Francisco, M., Kliebenstein, D.J. and Braam, J. (2013) Postharvest Circadian Entrainment Enhances Crop Pest Resistance and Phytochemical Cycling. Current Biology, 23, 1235–1241. https://doi.org/10.1016/j.cub.2013.05.034 McClung, C.R. (2011) The genetics of plant clocks. Burlington Academic Press, 74,105–139. https://doi.org/10.1016/B978-0-12-387690-4.00004-0 Endo, M., Shimizu, H., Nohales, M.A., Araki, T., & Kay, S.A. (2014) Tissue-specific clocks in Arabidopsis show asymmetric coupling. Nature. 515, 419–422. https://doi.org/10.1038/nature13919 Farmer, E. E. (2013) Surface-to-air signals. Nature, 411, 854–856. https://doi.org/10.1038/35081189 Ozawa, R., Arimura, G., Takabayashi, J., Shimoda, T. and Nishioka, T. (2000) Involvement of Jasmonate- and Salicylate-Related Signaling Pathways for the Production of Specific Herbivore-Induced Volatiles in Plants. Plant and Cell Physiology, 41(4), 391–398. https://doi.org/10.1093/pcp/41.4.391 De Moraes, C. M., Mescher, M. C. and Tumlinson, J. H. (2001) Caterpillar-induced nocturnal plant volatiles repel conspecific females. Nature, 410, 577–580. https://doi.org/10.1038/35069058 Yoneya, K. and Takabayashi, J. (2014) Plant–plant communication mediated by airborne signals: ecological and plant physiological perspectives. Plant Biotechnology, 31,409–416. https://doi.org/10.5511/plantbiotechnology.14.0827a Šimpraga, M., Takabayashi, J. and Holopainen, J. K. (2016) Language of plants: Where is the word? Journal of Integrative Plant Biology, 58, 343–349. https://doi.org/10.1111/jipb.12447 Pélissier, R., Violle, C. and Morel, J. B. (2021) Plant immunity: Good fences make good neighbors? Current Opinion in Plant Biology, 62, 102045. https://doi.org/10.1016/j.pbi.2021.102045 Marmolejo, L. O., Thompson, M. N. and Helms, A. M. (2021) Defense Suppression through Interplant Communication Depends on the Attacking Herbivore Species. Journal of Chemical Ecology, 47, 1049–1061. https://doi.org/10.1007/s10886-021-01314-6 Takagi, O., Sakamoto, M., Yoichi, H., Kokubo, H., Kawano, K. and Yamamoto, M. (2018) Discovery of Seasonal Dependence of Bio-Reaction Rhythm with Cucumbers. International Journal of Science and Research Methodology, 9, 163–175. https://www.researchgate.net/publication/331917254 Takagi, O., Sakamoto, M., Yoichi, H., Kokubo, H., Kawano, K. and Yamamoto, M. (2018) Relationship between Gas Concentration Emitted from Cut Cucumber Cross Sections and Growth Axis. International Journal of Science and Research Methodology, 9, 153–167. https://www.researchgate.net/publication/331917255 Takagi, O., Sakamoto, M., Kawano, K. and Yamamoto, M. (2022) Seasonal Changes in the Circadian Rhythm of Gas Released from Harvested Cucumbers. Natural Science, 14, 503–516. https://doi.org/10.4236/ns.2022.1411045 Kokubo, H., Koyama, S. and Takagi, O. (2010) Relationship between biophotons and gases generated from cucumber pieces. Journal of International Society of Life Information Science, 28, 84–94. https://doi.org/10.1016/j.pbiomolbio.2022.05.008 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3736017","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":259023425,"identity":"5963d4b0-a090-4ba6-8212-f06af30e848d","order_by":0,"name":"Osamu Takagi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYFACNiA2sGFgYGZgOECUBh6IljSStTAcJsFZ9uzHEj9+KTgvz8DO+/AAQ8U9uwaCtvCkHZaWMbht2MDMbnCA4UxxMmEtDOkN0hIGtxn3H2ZjOMDYlpBM0GE8/M+bf0sYnLNvYCZai0TaMckPBgcSYVrsCGu58SzNmsEgORmsJeFMQgJBLez9acY3f/yxs23gP8b84UNFgj1BLSDAzANjAa1IbCBGC+MPJA5xtoyCUTAKRsGIAgChQzZnjhGYbAAAAABJRU5ErkJggg==","orcid":"","institution":"International Research Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Osamu","middleName":"","lastName":"Takagi","suffix":""},{"id":259023426,"identity":"af5ab43a-4ac0-4fe4-8c3a-4852480e7f41","order_by":1,"name":"Masamichi Sakamoto","email":"","orcid":"","institution":"Aquavision Academy","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masamichi","middleName":"","lastName":"Sakamoto","suffix":""},{"id":259023427,"identity":"ffdc0107-66f2-4e13-9f31-45c1b6fb1bbe","order_by":2,"name":"Kimiko Kawano","email":"","orcid":"","institution":"International Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kimiko","middleName":"","lastName":"Kawano","suffix":""},{"id":259023428,"identity":"b763e1da-8559-417a-89d2-a8759a2fa8f0","order_by":3,"name":"Mikio Yamamoto","email":"","orcid":"","institution":"International Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mikio","middleName":"","lastName":"Yamamoto","suffix":""}],"badges":[],"createdAt":"2023-12-11 00:14:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3736017/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3736017/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":48228359,"identity":"2687b9c7-aac8-466d-a7ac-ce9ab464b3f9","added_by":"auto","created_at":"2023-12-14 18:17:40","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":782415,"visible":true,"origin":"","legend":"\u003cp\u003eSample preparation.\u003c/p\u003e\n\u003cp\u003eFig.1. (a) Cucumbers for sample preparation. (b) Samples A\u003csub\u003e1\u003c/sub\u003e, A\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e and B\u003csub\u003e2\u003c/sub\u003e. (c) Installation of samples A\u003csub\u003e1\u003c/sub\u003e, A\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e and B\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3736017/v1/6424a549918f1c7b63f84a5b.jpeg"},{"id":48228358,"identity":"2b912f15-00df-4d05-ba57-7db3d5f8c256","added_by":"auto","created_at":"2023-12-14 18:17:40","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":370272,"visible":true,"origin":"","legend":"\u003cp\u003eGas concentrations.\u003c/p\u003e\n\u003cp\u003eOne data-point in the figure represents the average of samples A\u003csub\u003e1\u003c/sub\u003e, A\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e and B\u003csub\u003e2\u003c/sub\u003e. Number of data n=468.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3736017/v1/10466de5e705ad691cf1bc05.jpeg"},{"id":48228356,"identity":"b96baca0-cd6a-407a-9bad-c96ab0fc337c","added_by":"auto","created_at":"2023-12-14 18:17:40","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":309177,"visible":true,"origin":"","legend":"\u003cp\u003eMoving average of the gas concentrations.\u003c/p\u003e\n\u003cp\u003eRed line: Weighted moving average of the gas concentration data shown in Fig.2, using the Gaussian function as weights. Blue line: Average of the gas concentration data (n=468), 353.8ppm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3736017/v1/00add9dd4d990ad97f1829ad.jpeg"},{"id":48605550,"identity":"37fa3b06-6224-479e-9b71-da0b1556ccf4","added_by":"auto","created_at":"2023-12-21 12:22:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":490423,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3736017/v1/74d621bf-c7c6-4115-9c5f-9ab2a685eccb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Do cucumbers sleep during Witching hour?","fulltext":[{"header":"One-Sentence Summary","content":"\u003cp\u003eEven cucumbers, which have no brain, sleep in the middle of the night.\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eFor many years, it was believed that sleep is a system controlled by the brain, and that animals without brains do not sleep. Recently, however, jellyfish and hydras have been shown to sleep, and several lines of evidence have been reported that even brainless organisms can sleep (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). There is also a Japanese proverb about the late night time from about 2:00 a.m. to 2:30 a.m. which coincides with the \u0026ldquo;witching hour\u0026rdquo;. The proverb states \"Kusa-Ki-Mo-Nemuru-Ushi-Mitsu-Doki\" which translates to \u0026ldquo;a time so quiet that not only people and animals but also grass and trees fall asleep\u0026rdquo;.\u003c/p\u003e \u003cp\u003eVegetables and fruits, which are plants, have been reported to have long-lasting biological responses after harvest (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) including the release of several types of volatiles when plants are exposed to stimuli or trauma after harvest (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The released volatiles in response to stimuli and trauma are believed to be plant-to-plant communications to elicit resistance against external enemies, to repair, and to promote immunological defenses. (\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe analyzed the concentrations of volatiles released from cucumber sections (\u003cem\u003eCucumis sativus\u003c/em\u003e) after harvest and reported that the circadian rhythm of gas concentrations varied with different seasons (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this paper, with the aim of answering the question of whether plants without brains also sleep, we analyzed the relationship between the diurnal time at which cucumber sections were prepared and the concentration of gas released from the sections within a designated period of time thereafter. As a result, we report that the gas concentration released from cucumber sections prepared at the \"Ushi-Mitsu-Doki\", or the Witching hour, was the lowest of the day.\u003c/p\u003e"},{"header":"SAMPLE PREPARATION AND GAS CONCENTRATION MEASUREMENT","content":"\u003cp\u003eFigures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a) and 1(b) show photographs of four uniform samples A\u003csub\u003e1\u003c/sub\u003e, A\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e, and B\u003csub\u003e2\u003c/sub\u003e prepared from four cucumbers. To prepare four uniform samples, we put one section from each of the four cucumbers into each Petri dish, for a total of four sections per dish. Here, A\u003csub\u003e1\u003c/sub\u003e and A\u003csub\u003e2\u003c/sub\u003e contain slices that share one cut surface which were positioned with that surface facing upwards. B\u003csub\u003e1\u003c/sub\u003e and B\u003csub\u003e2\u003c/sub\u003e are also similarly prepared, sharing single cut surfaces. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c) shows the Petri dishes placed in two layers after sample preparation. After leaving the Petri dishes as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c) for 30 minutes, the lids were removed from the Petri dishes, and the dishes containing the samples were placed in a sealed polypropylene container with a volume of 2.2 liters, and stored for 24 to 48 hours in a temperature-controlled (22 to 24 degrees Celsius) room with no direct sunlight. Although volatile components are released from the sample in the sealed container, the gas concentration reaches a maximum in about 12 hours, after which the measured gas concentration is found to remain in equilibrium (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). While the gas concentration remained at equilibrium, the concentration of hexanol gas was measured by aspirating 300 ml of gas from the sealed container using a gas detector tube (141L: GASTEC, Japan) and a gas detector device (GV-100: GASTEC).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMEASUREMENT AND ANALYSIS OF GAS CONCENTRATIONS\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the results of gas concentration measurements. The horizontal axis is the 24-hour period from 12:00 noon to 12:00 noon the next day. Here, the time represents the time when the four samples A\u003csub\u003e1\u003c/sub\u003e, A\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e, and B\u003csub\u003e2\u003c/sub\u003e were prepared. The vertical axis is gas concentration (ppm). In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e, 468 data points are plotted, each point being the average of the gas concentrations emitted from the four samples A\u003csub\u003e1\u003c/sub\u003e, A\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e, and B\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows in red the moving average of the gas concentrations shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. We used the weighted moving average with the Gaussian function as weights. Here, σ\u0026thinsp;=\u0026thinsp;0.5 hours was adopted for the calculation where σ\u003csup\u003e2\u003c/sup\u003e of the Gaussian function was used as the variance. The average of the 468 gas concentration data, 353.8 ppm, is indicated by the blue line.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the gas concentration emitted from the cucumber section varies depending on the time when the sample was prepared. Characteristically, the gas concentration reached a maximum around 15:00 and a minimum around 2:00 after midnight. This indicates that the gas production reaction is most active when cucumbers are cut around 15:00, gradually suppressed after 15:00, and most suppressed around 2:00 in a 24-hour day.\u003c/p\u003e \u003cp\u003eFrom these results the following 1 to 3 can be inferred.\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThat plant-to-plant communication becomes most active around 15:00. It appears that cucumber plants consider this time of day to be the most dangerous time and that communication and defenses need to be the most active around this time.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eCucumbers sense that around the 2:00 a.m. time, the need for inter-plant communication is at its lowest.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe increase in the moving average of gas concentrations from 2:00 to 6:00 indicates that the biological response is activated and this correlates with coming sunrise.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eWe sliced cucumbers and measured the gas concentrations of volatile components emitted from the cucumber sections. Analysis of the relationship between the time of cucumber cutting and gas concentration revealed that the intensity of the gas production reaction varied depending on the diurnal time when the cut was performed. In particular, the response was most active around 15:00 and most suppressed around 2:00.\u003c/p\u003e \u003cp\u003eWe concluded that cucumbers mimic a sleep-like state around 2:00, because this is the time when the gas production reaction of cucumbers is highly suppressed. In addition, since around two o\u0026rsquo;clock corresponds to the Japanese word \u0026ldquo;Ushi-Mitsu-Doki\u0026rdquo;, we proved that the Japanese proverb \"Kusa-Ki-Mo-Nemuru-Ushi-Mitsu-Doki\" which means that grass and trees fall asleep at Ushi-Mitsu-Doki is indeed correct.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e This research was funded by Science Peace Culture Foundation (SPC-F), (Chairman of the Board of Directors: Dr. Mikio Yamamoto).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eO.T. designed and performed the experiments. O.T. analyzed data and prepared the samples. O.T. wrote the paper. All authors discussed the experiments, analytical methods, and results and commented on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e Authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e All data are available in the main text or the supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRavi, D. N., Claire, N. B., Michael J. A., Ty, B., Justin, S. B., David, A. P., Paul, W. S., Viviana, G., Lea, G. (2017) The Jellyfish Cassiopea Exhibits a Sleep-like State. 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(2010) Relationship between biophotons and gases generated from cucumber pieces. Journal of International Society of Life Information Science, 28, 84\u0026ndash;94.\u003c/span\u003e \u003cspan\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pbiomolbio.2022.05.008\u003c/span\u003e\u003cspan address=\"10.1016/j.pbiomolbio.2022.05.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3736017/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3736017/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn recent years, several lines of evidence have been reported indicating that brainless organisms also sleep. In this paper, with the aim of answering the question of whether brainless plants also sleep, we analyzed the relationship between the diurnal time at which cucumber sections were prepared and the concentration of gas released from the sections within a certain period of time thereafter. The results showed that the gas concentration released from cucumber sections prepared around 2:00 a.m., the middle of the night, known in Japan as \"Ushi-Mitsu-Doki\", was the lowest of the day. Our results suggested that the gas production reaction of cucumbers changes depending on the time of sectioning, and that cucumbers were in a state resembling sleep at the \"Ushi-Mitsu-Doki\" time.\u003c/p\u003e","manuscriptTitle":"Do cucumbers sleep during Witching hour?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-14 18:17:35","doi":"10.21203/rs.3.rs-3736017/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"200cc282-f8ad-4c8b-a8da-1dfe96b4aa02","owner":[],"postedDate":"December 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":27350429,"name":"Biological sciences/Plant sciences"},{"id":27350430,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2023-12-21T12:14:29+00:00","versionOfRecord":[],"versionCreatedAt":"2023-12-14 18:17:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3736017","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3736017","identity":"rs-3736017","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}