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Food Chemistry: Molecular Sciences
Ultrasonic Treatment Suppresses Ethylene Signaling and Prolongs the Freshness ofSpinach
--Manuscript Draft--
Manuscript Number:
Full Title: Ultrasonic Treatment Suppresses Ethylene Signaling and Prolongs the Freshness ofSpinach
Short Title: Ultrasonic Treatment Prolongs Freshness of Spinach
Article Type: Full Length Article
Keywords: spinach, ultrasonication, stoma, abscisic acid, ethylene, ethylene-insensitive 3 bindingF-box protein
Corresponding Author: Shoji Oda, Ph.D.The University of Tokyo Graduate School of Frontier Sciences: Tokyo DaigakuDaigakuin Shinryoiki Sosei Kagaku KenkyukaKashiwa, Chiba JAPAN
Corresponding Author SecondaryInformation:
Corresponding Author's Institution: The University of Tokyo Graduate School of Frontier Sciences: Tokyo DaigakuDaigakuin Shinryoiki Sosei Kagaku Kenkyuka
Corresponding Author's SecondaryInstitution:
First Author: Shoji Oda, Ph.D.
First Author Secondary Information:
Order of Authors: Shoji Oda, Ph.D.
Masaaki Sakaguchi
Xiesong Yang
Qinyao Liu
Kohei Iwasaki
Kaori Nishibayashi
Order of Authors Secondary Information:
Abstract: There have been many studies investigating the application of ultrasonic treatment invegetables and fruits to eliminate surface contaminants including dirt, bacteria, andchemicals such as pesticides. Using a jet ultrasonic washer developed by us to washfood materials, we found that the freshness of leaf vegetables (spinach, komatsuna,Chinese cabbage (Shantung green leaf), cabbage, lettuce) was prolonged afterultrasonic treatment. The stomata of leaves were closed in the ultrasonicated spinach,whereas those in spinach soaked in water were open, resulting in water loss duringstorage. Transcriptome analysis revealed that the expression of Ethylene-insensitiveBinding F-box protein 1 and 2 (EBF1 and EBF2), which inhibit ethylene signaling, wasremarkably increased by ultrasonic treatment, strongly suggesting that the suppressionof ethylene signaling allowed stomatal closure in response to abscisic acid signals inthe ultrasonicated leaves. Although the precise mechanism of the induction of EBF1and EBF2 expression by ultrasonic treatment needs to be addressed in further studies,our findings suggest that ultrasonic treatment can be applied for the cleaning of leafvegetables as well as to prolong their freshness.
Suggested Reviewers: Catherine Barry-RyanDublin Institute of [email protected]
Hao Feng
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University of Illinois at [email protected]
Shunming WangAnhui Polytechnic [email protected]
Toshio SanoHosei [email protected]
Lixia ZhangResearch Institute of Agricultural Product Processing, Jiangsu Academy of [email protected]
Additional Information:
Question Response
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Cover letter
18th, December, 2020
Editor-in-Chief, Food Chemistry: Molecular Sciences,
Dear Dr. Siân Astley,
We are highly pleased to submit our original research article entitled “Ultrasonic Treatment
Suppresses Ethylene Signaling and Prolongs the Freshness of Spinach.” by Shoji Oda and the
others for the consideration for publication in Food Chemistry: Molecular Sciences.
We have newly designed a jet ultrasonic washer to wash vegetables and found that
freshness of leaf vegetables (spinach, komatsuna, Chinese cabbage (Shantung green leaf),
cabbage, lettuce) was retained for a long time after ultrasonic treatment. In this study, we
present evidences that ultrasonic treatment induced expression of Ethylene-insensitive 3
Binding F-box protein 1 and 2 (EBF1/2) and suppresses ethylene signals, allowing stoma close
during storage after the ultrasonic treatment. In contrast, in the spinach which was simply
soaked in water, stoma opens during storage and losses water to wilt faster than the
ultrasonicated spinach.
At first, we hypothesized that ultrasonic treatment might induce heat shock responses in
the vegetables; however, RNAseq results indicate that heat shock proteins are not induced after
the ultrasonic treatment. Of course, we have to agree that the precise molecular machinery of
the induction of the EBF1/2 expression by ultrasonic treatment should be addressed in further
studies, our findings suggest that ultrasonic treatment is applicable for cleaning leafy vegetables
as well as prolonging their freshness, and would have a revolutionary impact on the agriculture
and distribution industries.
I fortunately received the kind email from you to learn about your new Journal on 25th,
November, 2020. We were looking for the journal to submit this manuscript, since our study is at the
boundary between food science and plant physiology and we were afraid that our manuscript was
not acceptable to the journals in food science. Your timely email to us is a great luck and a great
honor for us to submit our manuscript to a new journal of future promising, Food Chemistry:
Molecular Sciences.
The manuscript has not been published elsewhere and is not being considered for publication
in another journal. All authors have read and approved submission of the manuscript. We added the
statements of conflicts of interest to disclose with the manuscript.
Cover Letter
We look forward to learning the results of peer review in due course.
Yours sincerely
Shoji Oda, Ph.D.,
Laboratory of Genome Stability,
Department of Integrated Biosciences,
Graduate School of Frontier Sciences,
The University of Tokyo
102 Life Science Building,
5-1-5 Kashiwanoha,
Kashiwa, Chiba, 277-8562, Japan
TEL:+81-4-7136-3671
FAX:+81-4-7136-3663
e-mail:[email protected]
Declaration of interests
☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Masaaki Sakaguchi and Shoji Oda are employed by The University of Tokyo, which owns the
Japan patent (JP6095087). Masaaki Sakaguchi owns the Japan patent (JP5863557 and JP6095087).
Masaaki Sakaguchi owns the Japan Utility Model Registration No. 3187858.
Conflict of Interest
Ultrasonic Treatment Suppresses Ethylene Signaling and Prolongs the Freshness of Spinach 1
2
Shoji ODA1*, Masaaki SAKAGUCHI1, Xiesong YANG1, Qinyao LIU1, Kohei IWASAKI2, Kaori 3
NISHIBAYASHI2 4
5
1 Department of Integrated Biosciences, Graduated School of Frontier Sciences, The University 6
of Tokyo 7
2 Kashiwanoha Urban Planning and Development Department/Venture Co-creation Department, 8
Mitsui Fudosan Co., Ltd. 9
10
* Corresponding author 11
TEL: +81-4-7136-3671 12
FAX: +81-4-7136-3669 13
Email: [email protected] 14
Title Page
1
Abstract 1
There have been many studies investigating the application of ultrasonic treatment in 2
vegetables and fruits to eliminate surface contaminants including dirt, bacteria, and chemicals 3
such as pesticides. Using a jet ultrasonic washer developed by us to wash food materials, we found 4
that the freshness of leaf vegetables (spinach, komatsuna, Chinese cabbage (Shantung green leaf), 5
cabbage, lettuce) was prolonged after ultrasonic treatment. The stomata of leaves were closed in 6
the ultrasonicated spinach, whereas those in spinach soaked in water were open, resulting in water 7
loss during storage. Transcriptome analysis revealed that the expression of Ethylene-insensitive 8
3 Binding F-box protein 1 and 2 (EBF1 and EBF2), which inhibit ethylene signaling, was 9
remarkably increased by ultrasonic treatment, strongly suggesting that the suppression of ethylene 10
signaling allowed stomatal closure in response to abscisic acid signals in the ultrasonicated leaves. 11
Although the precise mechanism of the induction of EBF1 and EBF2 expression by ultrasonic 12
treatment needs to be addressed in further studies, our findings suggest that ultrasonic treatment 13
can be applied for the cleaning of leaf vegetables as well as to prolong their freshness. 14
(177 words) 15
Key words: spinach, ultrasonication, stoma, abscisic acid, ethylene, ethylene-insensitive 3 16
binding F-box protein 17
18
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2
1. Introduction 19
It typically takes several hours or sometimes days for vegetables and fruits to be transported 20
from the farm to the grocery store, during which time these products pass through the distribution 21
system such as wholesale markets. In the case of leaf vegetables such as spinach, lettuce, or 22
cabbage, prior to shipment as commercial products, the roots are cut and the leaves begin to wilt 23
with the loss of the water supply upon harvest; further, they are exposed to severe water stress 24
(dry stress) during display in the grocery store. 25
Studies investigating the washing of vegetables and fruits with ultrasonic waves to remove 26
debris and other contaminants have been reported for decades (Lin & Epel., 1992; Zhou et al., 27
2012). To apply the cavitation effect for washing, it is necessary to soak the object requiring 28
cleaning in a liquid. In the case of vegetables, ultrasonic treatment is conducted using tap water 29
and detergents are occasionally added to facilitate washing; however, the use of detergents for 30
food materials should generally be avoided. 31
When harvested vegetables that are under water stress (dry stress) are soaked in water, they 32
resorb water and recover their freshness. We previously developed a novel jet ultrasonic washer 33
to clean food materials (Sakaguchi 2013, 2016; Sakaguchi & The University of Tokyo, 2017). In 34
addition to effects such as the elimination of pesticide residues, it was found that the freshness of 35
ultrasonicated spinach was prolonged compared to spinach soaked simply in water in a study of 36
3
the ultrasonication of leaf vegetables such as spinach and komatsuna. 37
To investigate the molecular mechanism of the recovery and freshness retention of 38
ultrasonically treated spinach following harvest, we focused on the behavior of stomata. During 39
storage, the stomata of spinach leaves soaked in water were open, while the stomata of 40
ultrasonicated spinach remained closed. It has been reported that heat shock reduces water loss in 41
fruits and vegetables to extend the shelf-life, and it has been proposed that the induction of heat 42
shock proteins could be involved in these protective effects (Saltveit, 1998; Baloch et al., 2006; 43
Rico et al., 2007). It is possible that the heat shock response is induced in vegetables subjected to 44
ultrasonic treatment, since the mechanical energy of the ultrasonic vibrations changes into thermal 45
energy due to the viscoelasticity of water. Transcriptome analysis in this study revealed that heat 46
shock proteins were not induced by ultrasonic treatment, while the expression of Ethylene-47
insensitive 3 Binging F-box 1 (EBF1) and EBF2, which suppress ethylene signaling, was 48
remarkably increased. It has been reported that ethylene signaling inhibits stomatal opening by 49
abscisic acid (She & Song, 2012; Tanaka et al., 2015). These findings strongly suggest that 50
ultrasonic treatment increased the expression of EBF1 and EBF2 and suppressed ethylene 51
signaling, allowing stomatal closure in response to abscisic acid in spinach during storage. As a 52
consequence, water loss was minimized and the freshness of the spinach was prolonged. 53
54
4
2. Materials & Methods 55
2.1. Ultrasonic treatment 56
We previously designed and produced a jet ultrasonic washer that was used for cleaning 57
food materials in this study. Details of this equipment are described in Utility Model Registration 58
of Japan No. 3187858 (Sakaguchi, 2013) and Japan Patents JP5863557 and JP6095087 (Sakaguchi, 59
2016; Sakaguchi & The University of Tokyo, 2017). The jet ultrasonic washer is equipped with a 60
cylindrical washing tank 200 mm in diameter, with a water depth of 120 mm. The power 61
consumption of the unit is 100 W. The washer is continuously supplied with fresh clean water 62
(tap water) from the water supply port (at the bottom center of the washing tank) at a flow velocity 63
of 0.5-1.0 L/min. The ultrasonic intensity (3-20 psi) was measured with the Sonic Meter SM-1000 64
(Shinka Industry Co., Ltd., Kawasaki, Japan). In this study, the washing tank was filled with tap 65
water and the vegetables were allowed to soak while being subjected to ultrasonic treatment at 40 66
kHz for 15 min. Then, the vegetables were removed from the washing tank, drained for 5 min and 67
kept in plastic containers at 4 °C or room temperature (25 °C). At the same time, vegetables were 68
soaked in tap water for 15 min without ultrasonic treatment as controls. 69
70
2.2. Vegetables 71
The vegetables used in the experiments (Japanese mustard spinach (komatsuna), lettuce, 72
5
cabbage and spinach) were purchased at a grocery store near Kashiwa Campus, The University 73
of Tokyo (Kashiwa City, Chiba Prefecture, Japan), and the freshest produce possible was chosen. 74
The freshness of vegetables is closely dependent on a variety of factors such as handling, 75
transportation and storage conditions, and time after harvest. Therefore, each produce sample was 76
divided into two parts; one part was washed with ultrasonic waves and the other was simply 77
soaked in water, thus controlling for quality differences among vegetables. Spinach, komatsuna 78
and Chinese cabbage (Shantung green leaf) grown by one of the authors (M.S.) at a private farm 79
were also used. 80
81
2.3. Transcriptome Analysis 82
Six bunches of spinach (Commercial product from Nagareyama City, Chiba Prefecture, 83
Japan) were washed with ultrasonic waves (40 kHz, 15 min) and jet, drained and kept in sealed 84
plastic containers at 4 °C in a refrigerator. After 3 or 24 h, one leaf with petiole was taken from 85
each bunch, immediately frozen between blocks of dry ice (1 kg), and powdered in a chilled 86
laboratory mortar with a pestle. A 1 g portion of the frozen powder was used for extraction of 87
total RNA (100-200 µg each) with TRIsure (Nihon Genetics, Tokyo, Japan). Total RNA (1 µg) 88
was subjected to RNAseq analysis with NovaSeq 6000 (illumina, San Diego, CA). The obtained 89
reads were mapped to the genome assembly of spinach in NCBI (ASM200726v1, 90
6
https://www.ncbi.nlm.nih.gov/assembly/GCF_002007265.1) and gene annotation was conducted 91
with Spinacia oleracea Annotation Release 100 by NCBI. Six bunches of spinach soaked in water 92
for 15 min without ultrasonic treatment were subjected to extraction of total RNA and analyzed 93
in the same manner as above. In this study, RNAseq analysis was conducted once for each RNA 94
sample extracted from control, ultrasonicated or simply soaked spinach leaves at 3 and 24 h after 95
treatment. Fold changes (FC) were calculated using edgeR (Robinson et al., 2010) and genes with 96
FC >1.5 were selected as differentially expressed genes (DEGs). The BioProject ID is 97
PRJDB10558 and the DRA accession ID is DRA010883. The accession number of each read is 98
DRR248963, DRR248964, DRR248965, DRR248966, DRR248967 for the control, 3 h after 99
water soaking, 24 h after water soaking, 3 h after ultrasonication, and 24 h after ultrasonication, 100
respectively. 101
102
https://www.ncbi.nlm.nih.gov/assembly/GCF_002007265.1
7
3. Results 103
3.1. Ultrasonic washing recovers and prolongs the freshness of leaf vegetables compared to 104
soaking in water 105
Vegetables (spinach, komatsuna, Chinese cabbage (Shantung green leaf), cabbage, lettuce) 106
were washed with ultrasonic waves for 10-15 min and then stored in sealed plastic containers to 107
prevent desiccation in a refrigerator for 2-24 h. The leaves were observed to spread widely and 108
showed less wilt than those soaked in water only (Fig. 1 A, B). The leaves appeared fresh and 109
recovered their crispy texture. Since it is well known that harvested vegetables recover their 110
texture and freshness when soaked in water, we considered the possibility that the ultrasonic 111
treatment activated water absorption of vegetables. Since it was very difficult to completely drain 112
the water from the leaf surface of the vegetables following treatment, we could not accurately 113
measure the change in wet weight of the ultrasonicated vegetables. Thus, we measured the change 114
in the wet weight of the petioles of spinach leaves after ultrasonic treatment. When petioles were 115
soaked in water, their wet weight increased and water absorption was confirmed. The wet weight 116
of the petioles treated with ultrasonic waves also increased, indicating that water was absorbed as 117
well. Subsequently, the petioles were kept in a sealed plastic container and changes in wet weight 118
were observed for 25 days (Fig. 2A). The wet weight of the ultrasonicated petioles decreased at 119
the same rate as the untreated petioles (not soaked in water), whereas the petioles soaked in water 120
8
showed a more rapid decrease in wet weight and wilted (Fig. 2B). 121
122
3.2. Ultrasonic treatment results in stomatal closure and avoidance of water loss in vegetables 123
We hypothesized that ultrasonic treatment regulates the opening/closing of stomata in 124
vegetables. Spinach leaves were soaked in water or washed with ultrasonic waves for 15 min. 125
Subsequently, the leaves were kept in sealed containers in the refrigerator for 3 h and the stomata 126
were microscopically observed. Almost all stomata were open in both the sample soaked into 127
water only and that treated with ultrasonic waves. When the leaves were left for 21 h at 4oC in a 128
sealed container and again examined microscopically, it was found that approximately half of the 129
stomata were open in the soaked leaves (Fig. 3A, C), while almost all stomata were closed in the 130
leaves ultrasonically treated (Fig. 3B, D). The stomata closure by the ultrasonic treatment was 131
also observed in komatsuna leaves. 132
133
3.3. Transcriptome analysis revealed EBF1 and EBF2 were induced in ultrasonicated leaves 134
To verify the contribution of heat shock proteins in "refreshing" the ultrasonicated spinach, 135
we conducted transcriptome analysis and investigated the expression of 19,228 genes in spinach. 136
It was found that most genes were expressed similarly in the ultrasonicated and water-soaked 137
leaves (Fig. 4). At 3 h after treatment, the expression of 51 genes was decreased in the leaves 138
9
soaked in water and increased in the leaves subjected to ultrasonic treatment (Fig. 4A) 139
(Supplementary Table 1). Further, the expression of 42 genes was increased in the water-soaked 140
samples and decreased in the ultrasonically treated samples (Fig. 4A) (Supplementary Table 2). 141
At 24 h after treatment, the expression of 44 genes was decreased in the leaves soaked in water 142
and increased in the leaves ultrasonically treated (Fig. 4B) (Supplementary Table 3), and the 143
expression of 48 genes was increased in the water-soaked leaves and decreased in the 144
ultrasonicated leaves (Fig. 4B) (Supplementary Table 4). No heat shock proteins were identified 145
in the genes showing increased expression in the ultrasonicated leaves in this study. 146
It is well known that abscisic acid and ethylene control the opening and closing of stomata 147
(Zhu, 2002; She & Song, 2012; Tanaka et al., 2015). Thus, we analyzed the gene expression related 148
to these plant hormones in detail. In transcriptome analysis of the spinach leaves 3 h after soaking 149
in water, the gene expression of abscisic acid synthesizing enzyme (LOC110805680) was 150
decreased and that of abscisic acid degrading enzyme (LOC110788345) was increased, indicating 151
the inactivation of abscisic acid signaling. These changes were similar to those in the 152
ultrasonicated leaves (Table 1). It is possible that the spinach leaves resorb water when soaked or 153
ultrasonicated, and abscisic acid signaling might be weakened because of the mitigated water 154
stress. In the leaves soaked in water and stored for 24 h, gene expression of both abscisic acid 155
synthesizing enzyme (LOC110805680) and abscisic acid degrading enzyme (LOC110788345) 156
10
showed further decreases. In the ultrasonicated leaves stored for 24 h, gene expression of abscisic 157
acid degrading enzyme (LOC110788345) decreased as well, while that of abscisic acid 158
synthesizing enzyme (LOC110805680) increased, suggesting that abscisic acid signaling was re-159
activated 24 h after the ultrasonic treatment. RAB18 (LOC110776340) is one of the stress-160
responsive genes encoding the glycine-rich dehydrin protein that is expressed in the guard cells 161
of stomata, and its expression is induced by abscisic acid (Nylander et al., 2001; Tanaka et al., 162
2005). Expression of RAB18 decreased 3 h after treatment and increased 24 h after treatment in 163
both the soaked and the ultrasonicated leaves (Table 1), strongly suggesting that the leaves were 164
again under water stress after 24-h storage and reactivation of abscisic signaling (Supplementary 165
Table 5). 166
Next, we focused on the expression of genes involved in ethylene synthesis (Table 1, 167
Supplementary Table 6). Gene expression of ACC synthase (LOC110805602, LOC110805592) 168
and ACC oxidase (LOC110777334, LOC110803795) (Johnson & Ecker, 1998; Yang, 2003) 169
increased 3 h after treatment in both the soaked and the ultrasonicated leaves. Expression of ACC 170
synthase (LOC110805602, LOC110805592) decreased 24 h after treatment, but expression of 171
ACC oxidase (LOC110777334, LOC110803795) was maintained in both the soaked and the 172
ultrasonicated leaves (Table 1); thus, ultrasonic treatment did not affect the expression of ACC 173
synthase (LOC110805602, LOC110805592) and ACC oxidase (LOC110795784, 174
11
LOC110777334, LOC110803795). Interestingly, Ethylene-insensitive 3 Binding F-box protein 1 175
(EBF1, LOC110788382) was identified as a DEG with a large read count, and its expression was 176
decreased in the soaked leaves but increased in the ultrasonicated leaves 3 h after the treatment. 177
Then, the expression of EBF1 was remarkably increased in the ultrasonicated leaves 24 h after 178
the treatment. The expression of Ethylene-insensitive 3 Binding F-box protein 2 (EBF2, 179
LOC110796826) was increased 3 h after the treatment and remarkably increased 24 h after the 180
treatment both in the soaked and ultrasonicated leaves (Table 1). 181
Gene expression related to the other major plant hormones, cytokinin, gibberellin and auxin 182
was similar in the soaked and ultrasonicated leaves (Supplementary Tables 7, 8, 9). No noteworthy 183
changes associated with the ultrasonic treatment were observed for p450 gene expression 184
(Supplementary Table 10), which includes the gibberellin synthesizing pathway genes (Pimenta 185
Lange & Lange, 2006), suggesting that these were not impacted by ultrasonic treatment. 186
187
188
4. Discussion 189
Using a jet ultrasonic washer we have developed, the freshness of spinach leaves was 190
recovered and prolonged after washing with ultrasonic waves. Heat shock treatment has been 191
widely applied to maintain the freshness and extend the shelf-life of harvested fresh vegetables 192
12
and fruits, as heat shock proteins might be induced and protective (Saltveit, 1998; Baloch et al., 193
2006; Rico et a., 2007). At first, we hypothesized that ultrasonic washing of vegetables could heat 194
the vegetables and induce heat shock proteins; however, the transcriptome analysis indicated that 195
the expression of heat shock proteins was not promoted in the ultrasonicated spinach. 196
Post-harvest vegetables undergo water stress and the abscisic acid pathway is strongly 197
activated (Bray, 1997; Zhu, 2002). The activation of abscisic acid degrading enzyme in the leaves 198
3 h after water soaking indicates the mitigation of water stress. Gene expression related to 199
ethylene synthesis (Yang & Hoffman, 1984; Kevin et al., 2002; Yang, 2003) showed no 200
remarkable changes during storage, suggesting that a small amount of ethylene might be 201
synthesized continuously during 24-h storage after treatment. Expression of RAB18 was 202
remarkably increased in both the soaked and the ultrasonicated leaves. Since RAB18 expression 203
is strongly dependent on abscisic acid signaling and is used as an index of active abscisic acid 204
signaling (Nylander et al., 2001; Tanaka et al., 2015), we can assume that abscisic acid signaling 205
is again activated during 24-h storage after soaking in water. The expression of abscisic acid 206
synthase increased in the ultrasonicated leaves after 24-h storage. In contrast, soaked leaves 207
showed decreased expression of abscisic acid synthase after 24-h storage, suggesting that abscisic 208
acid signaling was less activated in the water-soaked leaves. 209
In the ultrasonicated leaves, EBF1 and EBF2 gene expression increased remarkably during 210
13
storage after treatment (Table 1). Ethylene is one of the major plant hormones that controls 211
germination, flower bud formation, fruit maturation or plant aging (Khan et al., 2017; Dubois et 212
al., 2018), and EBF1 and EBF2 strictly regulate the physiological actions of ethylene (Guo & 213
Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004; Yang et al., 2010). Both EBF1 and EBF2 214
bind EIN3, which is the major transducer in ethylene signaling, resulting in EIN3 ubiquitinization 215
and subsequent proteolyzation by the proteasome system (Guo & Ecker, 2003; Potuschak et al., 216
2003; Gagne et al., 2004). The weak expression of EBF1 and EBF2 in the soaked leaves may 217
result in quality deterioration such as ethylene-induced aging, in addition to water loss. EBF2 218
expression is reported to be induced by ethylene in Arabidopsis (Potuschak et al., 2003; Konish 219
& Yanagisawa, 2008); however, the mechanism of induced expression of EBF1 has not yet been 220
elucidated. Although there is overlap in the functions of EBF1 and EBF2, EBF1 responds to lower 221
concentrations of ethylene, while EBF2 responds to higher concentrations (Binder et al., 2007). 222
In the soaked spinach, expression of EBF1 and EBF2 was weak and the continuous production of 223
ethylene might inhibit stomata closure by abscisic acid. It is known that harvested vegetables 224
resorb water and temporarily recover their freshness when soaked in water, but subsequently 225
quickly lose water and wilt. The above findings suggest the involvement of ethylene in this well-226
known phenomenon. 227
Various antagonistic or reciprocal physiological interactions are known between abscisic 228
14
acid and ethylene signaling, which are known to function antagonistically in controlling stomatal 229
opening and closing (Beaudoin et al., 2000; Schroeder et al., 2001; Dodd, 2003; Li & Huang, 2011). 230
Abscisic acid signaling is activated by the lack of water to promote stomatal closure, while 231
ethylene has inhibitory effects (She & Song, 2012; Tanaka et al., 2015). In this study, we observed 232
that ultrasonic treatment remarkably increased the expression of EBF1 and EBF2. It is proposed 233
that ultrasonic treatment suppresses ethylene signaling via EBF1 and EBF2 and allows stomatal 234
closure by abscisic acid, thereby avoiding water loss through the stomata (Schroeder et al., 2001; 235
Dodd, 2003). In contrast, the weak expression of EBF1 and EBF2 may allow ethylene to prevent 236
stomatal closure by abscisic acid (She & Song, 2012; Tanaka et al., 2015). In this scenario, the 237
spinach leaf might rapidly lose water after water soaking and wilt, in addition to the aging 238
promoting action of ethylene. 239
The molecular mechanism of the promotion of EBF1 and EBF2 expression by ultrasonic 240
treatment needs to be elucidated in future studies. It is acknowledged that low-frequency vibration 241
promotes ethylene synthesis and affects the physiological conditions of plants (Telewski & Jaffe, 242
1986). In recent years, evidence that ultrasonic treatment has physiological effects on plants has 243
been accumulating (Yu et al., 2016; Ding et al., 2017, 2018; Wang et al., 2019). Specifically, it 244
has been reported that ultrasonic treatment promotes germination and increases -aminobutyric 245
acid (GABA) content (Yang et al., 2015, Ding et al., 2017, 2018). It is highly likely that ultrasonic 246
15
treatment promotes water resorption and modulates ethylene and abscisic acid signaling in seeds, 247
both of which promote germination in many plant species (Johnson & Ecker, 1998; Vishal & 248
Kumar, 2018). 249
Since the spinach genome remains poorly characterized, it was not possible to conduct 250
detailed transcriptome analysis to elucidate the molecular mechanisms of the biological effects of 251
ultrasonic treatment. Thus, detailed molecular biological analysis in a well-studied model plant 252
such as Arabidopsis could assist in elucidating the precise molecular events induced by ultrasonic 253
treatment in plants. In human, ultrasound has been used as a therapeutic tool to stimulate 254
osteoblast differentiation (Macione et al., 2015); however, the precise mechanisms of the 255
biological effects of ultrasound are almost completely unknown. In life science laboratories, high-256
intensity ultrasonic treatment is widely used to disrupt cells or tissues in preparing samples for 257
biochemical experiments. On the other hand, the effects of low-intensity ultrasonic treatment on 258
living cells and tissues have not been investigated, representing a new frontier for future 259
exploration. 260
It is likely that the freshness of vegetables can be prolonged by ultrasound-induced 261
inhibition of the effects of ethylene, without the need for chemical reagents, making it a very 262
desirable for food applications. We developed the jet ultrasonic washer to remove contaminants 263
such as dirt, chemicals and radioactive compounds from the surfaces of vegetables. The soaking 264
16
in water of vegetables, which are harvested and transported over hours or days, is known to 265
recover their freshness; however, wilting occurs quickly thereafter, reducing the commercial value 266
of products. Hence, harvested and transported vegetables should not be soaked in water. In 267
contrast, ultrasonic treatment produces two favorable outcomes: the thorough washing of 268
vegetables and fruits, and rehydration to recover and prolong freshness. We have preliminary 269
confirmed that some commercially available ultrasonic washers (without jet) can prolong 270
freshness of spinach and other leaf vegetables. It is anticipated that the findings of this study will 271
facilitate the distribution of fresh vegetables and fruits, and provide significant benefits to the 272
agricultural sector, distributors and consumers alike. 273
274
5. Conclusions 275
The freshness of leaf vegetables (spinach, komatsuna, Chinese cabbage (Shantung green 276
leaf), cabbage, lettuce) washed using a jet ultrasonic washer was recovered and prolonged after 277
ultrasonic treatment. Transcriptome analysis revealed that ultrasonic treatment induced 278
expression of Ethylene-insensitive Binding F-box protein 1 and 2 (EBF1 and EBF2) in spinach 279
leaves and suppressed ethylene signaling. During storage after ultrasonic treatment, spinach 280
leaves exhibited water stress and abscisic acid signaling was activated to close stomata and 281
prevent water loss. In contrast, in water-soaked leaves, the expression of EBF1 and EBF2 was 282
17
weak and the stomata did not close, resulting in the rapid loss of water and wilting than 283
ultrasonicated leaves during storage. Although the precise mechanism of the induction of EBF1 284
and EBF2 expression by ultrasonic treatment should be addressed in further studies, our findings 285
suggest that ultrasonic treatment is applicable for cleaning leafy vegetables as well as prolonging 286
their freshness. 287
288
289
Author contributions 290
Shoji ODA: Conceptualization, Investigation, Data curation, Writing - Original Draft, 291
Writing - Review & Editing, Supervision. Masaaki SAKAGUCHI: Conceptualization, 292
Resources, Methodology, Investigation, Project administration. Xiesong YANG: 293
Investigation. Qinyao LIU: Investigation. Kohei IWASAKI: Project administration, 294
Writing - Original Draft. Kaori NISHIBAYASHI: Conceptualization, Writing - Original 295
Draft, Supervision. 296
297
Acknowledgements 298
The authors thank Dr. Misato Ohtani for her critical reading of the manuscript and helpful 299
suggestions, Mr. Ken-ichi Saito for his support, and Ms. Yukie Oda-Kato for her 300
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assistance. 301
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Fig. 1. Effect of ultrasonic treatment on leaf vegetable wilting.
A) Komatsuna (Japanese mustard spinach) was washed with ultrasonic waves (40
kHz) (left) or soaked in water (right) for 15 min, then drained and stored for about
2 h. Ultrasonically treated komatsuna exhibited leaf spread, while komatsuna
soaked in water began to wilt. B) Chinese cabbages (Shantung green leaf) were
washed with ultrasonic waves (left) or soaked in water (right), then drained and
stored for about 2 h. Ultrasonic treatment prevented wilting of Shantung green
leaf.
Ultrasonicated Soaked in water
A
B
Ultrasonicated Soaked in water
Figure1 Click here to access/download;Figure;Figure_1.pptx
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A
B
Ultrasonicated_No1
Ultrasonicated_No2
No Treatment_No1
No Treatment_No2
Soaked in water_No1
Soaked in water_No2
Time of storage (day)
Dec
reas
e in
wet
wei
ght
(%)
Soaked in water
No Treatment
Ultrasonicated
Fig. 2. Effect of ultrasonic treatment on changes in the wet weight of spinach.
A) The petioles of spinach were ultrasonicated or soaked in water for 15 min, then
placed in a sealed plastic container and stored in a refrigerator. Prior to storage, any
surface water was removed by wiping with a paper towel. B) Changes in wet weight
of petioles. The wet weight of spinach petioles immediately after soaking in water or
ultrasonic treatment was assumed to be 100%, and the decrease in wet weight is
shown.
Figure2 Click here to access/download;Figure;Figure_2.pptx
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A
C
B UltraonicatedSoaked in water
Fig. 3. Effect of ultrasonic treatment on stomatal opening in spinach leaves.
A, B) Spinach leaves were ultrasonicated or soaked in water for 15 min, and after thorough
draining of any remaining water, the leaves were placed in a sealed plastic container and
stored in a refrigerator for 21 h. Stomata on the dorsal surface of leaves were observed
under a microscope. Bars represent 50 mm. C, D) The width and the length of the stomatal
aperture were measured and stomatal aperture index was calculated by division of the
width through the length of the aperture. Histograms of stomatal aperture index in the
soaked leaves and the ultrasonicated leaves were presented. Parenthesized figure in C and
D is an open stoma in the soaked leaf and a closed stoma in the ultrasonicated leaf,
respectively.
0
2
4
6
8
10
12
14
Nu
mb
er o
f st
om
ata
0
1
2
3
4
5
6
7
8
9
14
10
12
8
6
4
2
0
8
4
6
2
1
0
Nu
mb
er o
f st
om
ata
Stomatal aperture index
0.2 0.3 0.4 0.5 0.70.6
Stomatal aperture index
0.2 0.3 0.4 0.5 0.70.6
D
Figure3 Click here to access/download;Figure;Figure_3.pptx
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Down in SonicatedNo change
in sonicatedUp in sonicated
Dow in Soaked 835 546 51
No change in soaked 1086 13,471 881
Up in soaked 42 698 1,600
After 3 hours storage
Down in sonicatedNo change in sonicated
Up in sonicated
Down in Soaked 2,776 887 44
No change in soaked 856 10,403 671
Up in soaked 48 723 2,820
After 24 hours storage
Total gene = 19,228
A
B
Total gene = 19,228
Fig. 4. Effect of ultrasonic treatment on gene expression in spinach.
A) Among the 19,228 genes examined, 51 genes showed decreased expression in the
sample soaked in water and increased expression in the ultrasonicated sample; and 42
genes showed increased expression in the sample soaked in water and decreased
expression in the ultrasonicated sample 3 h after the treatment. There were no
differences in the expression of the remaining 19,135 genes between the samples soaked
in water and the ultrasonicated samples. B) Twenty-four hours after the treatment, 44
genes showed decreased expression in the sample soaked in water and increased
expression in the ultrasonicated sample. Further, the expression of 48 genes increased in
the water-soaked sample and decreased in the ultrasonicated sample. There were no
differences in the expression of the remaining 19,136 genes between the sample soaked
in water and the ultrasonicated sample.
Figure4 Click here to access/download;Figure;Figure_4.pptx
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Graphical abstract Click here to access/download;Figure;GraphicAbstract.tif
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Table 1.
Gene_Symbol Description DIP_3H/Control.fc SONIC_3H/Control.fc DIP_24H/Control.fc SONIC_24H/Control.fc SONIC_3H/DIP_3H.fc SONIC_24H/DIP_24H.fc Control_Read_Count DIP_3H_Read_Count SONIC_3H_Read_Count DIP_24H_Read_Count SONIC_24H_Read_Count
Abscisic acid synthesizing enzyme
LOC110805680 9-cis-epoxycarotenoid dioxygenase NCED1, chloroplastic -3.605413667 -4.247305732 -8.129044247 -4.210707956 -1.178037406 1.93057618 9245 2979 2399 1322 2701
Abscisic acid degrading enzyme
LOC110788345 abscisic acid 8'-hydroxylase 1-like 1.572890484 1.691034225 -1.721223308 -1.832461377 1.075113029 -1.064626772 3210 5866 5983 2168 2155
LOC110776340 dehydrin Rab18-like -1.461261511 -1.639375025 4.560104889 4.576129717 -1.121891151 1.003513399 3723 2960 2503 19737 20960
ACC synthase
LOC110805602 1-aminocyclopropane-1-carboxylate synthase-like 2.271920338 1.812485522 -4.984110472 -2.592415929 -1.2534828 1.922601536 2217 5852 4429 517 1052
LOC110805592 1-aminocyclopropane-1-carboxylate synthase-like 2.246692241 1.778404764 -5.312201415 -2.579144184 -1.263318373 2.059709139 2235 5834 4381 489 1066
ACC oxidase
LOC110795784 1-aminocyclopropane-1-carboxylate oxidase homolog 4-like 1.014762454 1.161219685 -1.127251131 1.008256821 1.144326701 1.136558755 14892 17557 19060 15358 18472
LOC110777334 1-aminocyclopropane-1-carboxylate oxidase-like 1.56267106 1.591448564 1.94422602 2.192844766 1.01841568 1.127875332 13915 25263 24408 31451 37539
LOC110803795 1-aminocyclopropane-1-carboxylate oxidase 4-like 1.81208683 1.829860724 1.814747817 1.5096348 1.009808577 -1.202110587 27713 58344 55893 58466 51469
LOC110788382 EIN3-binding F-box protein 1-like (EBF1) -1.58442855 1.841045792 1.43413969 2.634585749 2.917010479 1.837050214 4075 2988 8269 6794 13208
LOC110796826 EIN3-binding F-box protein 2-like (EBF2) 1.619344413 10.63233002 10.13415665 18.32485253 6.565940378 1.808210765 321 604 3763 3783 7239
Gene symbol: Gene ID in the NCBI database (https://www.ncbi.nlm.nih.gov/); DIP_3H/Control.fc: fold change of the soaked leaves 3 h after the treatment against control (no treatment); SONIC_3H/Control.fc: fold change of the ultrasonicated leaves 3 h after the treatment against control (no treatment);
DIP_24H/Control.fc: fold change of the soaked leaves 24 h after the treatment against control (no treatment); SONIC_24H/Control.fc: fold change of the ultrasonicated leaves 24 h after the treatment against control (no treatment); SONIC_3H/DIP_3H.fc: fold change of the ultrasonicated leaves 3 h after the treatment
against the soaked leaves; SONIC_24H/DIP_24H.fc: fold change of the ultrasonicated leaves 24 h after the treatment against the soaked leaves; Control_Read_Count: Read counts in the not treated leaves (control); DIP_3H_Read_Count: Read counts in the soaked leaves 3 h after the treatment;
SONIC_3H_Read_Count: Read counts in the ultrasonicated leaves 3 h after the treatment; DIP_24H_Read_Count: Read counts in the soaked leaves 24 h after the treatment; SONIC_24H_Read_Count: Read counts in the ultrasonicated leaves 24 h after the treatment.
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Highlights
Ultrasonicated spinach retains water for a longer time than spinach soaked in water. (84
characters)
Ultrasonication induces stomatal closure in spinach leaves. (60 characters)
Ultrasonication stimulates EBF1/EBF2 expression and suppresses ethylene signaling. (82
characters)
Highlights
Supplementary Table1
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Supplementary Table8
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