food chemistry: molecular sciences...cabbage, lettuce) was retained for a long time after ultrasonic...

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

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation

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

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation

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

Manuscript Click here to view linked References

https://www.editorialmanager.com/fochms/viewRCResults.aspx?pdf=1&docID=156&rev=0&fileID=3318&msid=15b5d98e-856a-47fa-8502-e207d74eec9chttps://www.editorialmanager.com/fochms/viewRCResults.aspx?pdf=1&docID=156&rev=0&fileID=3318&msid=15b5d98e-856a-47fa-8502-e207d74eec9c

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

18

assistance. 301

References 302

Beaudoin, N., Serizet, C., Gosti, F. & Giraudat J. (2000). Interactions between Abscisic 303

Acid and Ethylene Signaling Cascades. The Plant Cell, 12, 1103-1115. 304

https://doi.org/10.1105/tpc.12.7.1103. 305

306

Baloch, M. S., Morimoto, T. & Hatou, K. (2006). A heat stress application technique for 307

reducing water loss of fruits during storage. Environmental Control in Biology, 44, 31-40. 308

https://doi.org/10.2525/ecb.44.31. 309

310

Binder, B. M., Walker, J. M., b, Gagne, J. M., Emborg, T. J., Hemmann, G., Bleecker, A. 311

B. & Vierstrab, R. D. (2007). The Arabidopsis EIN3 Binding F-Box Proteins EBF1 and 312

EBF2 Have Distinct but Overlapping Roles in Ethylene Signaling. The Plant Cell, 19, 313

509–523. https://doi.org/10.1105/tpc.106.048140. 314

315

Bray, E.A. (1997). Plant responses to water deficit. Trends in plant science, 2, 48-54. 316

https://doi.org/10.1016/S1360-1385(97)82562-9. 317

318

19

Ding, J., Ulanov, A.V., Dong, M., Yang, T., Nemzer, B. V., Xiong, S., Zhao, S. & Feng, 319

H. (2017). Enhancement of gama-aminobutyric acid (GABA) and other health-related 320

metabolites in germinated red rice (Oryza sativa L.) by ultrasonication. Ultrasonics-321

Sonochemistry, 40, 791-797. doi: 10.1016/j.ultsonch.2017.08.029. 322

323

Ding, J., Hou, G.G., Nemzer, B. V., Xiong, S., Dubate, A., & Feng, H. (2018). Effects of 324

controlled germination on selected physicochemical and functional properties of whole-325

wheat flour and enhanced γ-aminobutyric acid accumulation by ultrasonication. Food 326

Chemistry, 243, 214-221. https://doi.org/10.1016/j.foodchem.2017.09.128. 327

328

Dodd, I. C. (2003). Hormonal interactions and stomatal responses. Journal of Plant 329

Growth Regulation, 22, 32-46. doi: 10.1007/s00344-003-0023-x 330

331

Dubois, M. Van den Broeck, L. & Inzé, D. (2018). The Pivotal Role of Ethylene in Plant 332

Growth. Trends in Plant Science, 23, 311-323.https:// 333

doi.org/10.1016/j.tplants.2018.01.003 334

335

Gagne, J.M., Smalle, J., Gingerich, D.J., Walker, J. M., Yoo, S. D., Yanagisawa, S. & 336

20

Vierstra, R.D. (2004). Arabidopsis EIN3-binding F-box 1 and 2 form ubiquitin-protein 337

ligases that repress ethylene action and promote growth by directing EIN3 degradation. 338

Proceedings of the National Academy of Sciences USA, 101, 6803-6808. 339

https://doi.org/10.1073/pnas.0401698101. 340

341

Guo, H. & Ecker, J. R. (2003). Plant Responses to Ethylene Gas Are Mediated by 342

SCFEBF1/EBF2-Dependent Proteolysis of EIN3 Transcription Factor. Cell, 115, 667-677. 343

https://doi.org/10.1016/S0092-8674(03)00969-3. 344

345

Johnson, P. R. & Ecker, J. R. (1998). The ethylene gas signal transduction pathway: a 346

molecular perspective. Annual Review of Genetics, 32, 227-254. 347

https://doi.org/10.1146/annurev.genet.32.1.227. 348

349

Khan, N. A., M. I. R. Khan, M. I. R., Ferrante, A. & Poor, P. (2017). Ethylene: A key 350

regulatory molecule in plants. Frontiers in Plant Science, 8, 1782. 351

https://doi.org/10.3389/fpls.2017.01782. 352

353

Kevin L.-C. Wang, K. L. C., Li, H & Ecker, J. R. (2002). Ethylene Biosynthesis and 354

21

Signaling Networks. The Plant Cell, S131-S151. https://doi.org/10.1105/tpc.001768. 355

356

Konishi, M. and Yanagisawa, Y. (2008). Ethylene signaling in Arabidopsis involves 357

feedback regulation via the elaborate control of EBF2 expression by EIN3. The Plant 358

Journal, 55, 821-831. doi: 10.1111/j.1365-313X.2008.03551.x 359

360

Li, Z. & Huang, R. (2011). The reciprocal regulation of abscisic acid and ethylene 361

biosyntheses. Plant Signaling and Behavior, 6, 1647-1650. 362

https://doi.org/10.4161/psb.6.11.17756. 363

364

Lin, L. & Epel, D (1992). Dynamic ultrasonic cleaning and disinfecting device and 365

method, United States Patent 5,113,881. 366

367

Macione, J., Long, D., Nesbitt, S., Wentzell, S., Yokota, H., Pandit, V. & Kotha, S. (2015). 368

Stimulation of osteoblast differentiation with guided ultrasound waves. Journal of 369

Therapeutic Ultrasound. 3, 12-18. doi: 10.1186/s40349-015-0034-7. 370

371

Nylander, M., Svensson, J., Palva, E. T., & Welin, B. V. (2001). Stress-induced 372

22

accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana. Plant 373

Molecular Biology, 45, 263–279. doi: 10.1023/a:1006469128280. 374

375

Potuschak, T., Lechner, E., Parmentier, Y., Yanagisawa, S., Grava, S., Koncz, C. & 376

Genschik, P. (2003). EIN3-Dependent Regulation of Plant Ethylene Hormone Signaling 377

by Two Arabidopsis F Box Proteins: EBF1 and EBF2. Cell, 115, 679-689. DOI: 378

10.1016/s0092-8674(03)00968-1. 379

380

Rico, D., Martín-Diana, A. B., Barat, J. M. & Barry-Ryana, C. (2007). Extending and 381

measuring the quality of fresh-cut fruit and vegetables: a review. Trends in Food Science 382

& Technology, 18, 373-386. https://doi.org/10.1016/j.tifs.2007.03.011. 383

384

Robinson, M.D., McCarthy, D.J. & Smyth, G.K. (2021). edgeR: a Bioconductor package 385

for differential expression analysis of digital gene expression data. Bioinformatics, 26, 386

139-140. DOI: 10.1093/bioinformatics/btp616 387

388

Sakaguchi M. 2013. Ultrasonic washer. Japan Utility Model Registration No. 3187858. 389

390

23

Sakaguchi M. 2016. Ultrasonic washer. Japan patent JP5863557. 391

392

Sakaguchi M. & The University of Tokyo. 2017. Ultrasonic washer. and Sonication 393

method. Japan patent JP6095087. 394

395

Saltveit, M. E. (1998). Heat-Shock and Fresh Cut Lettuce. Perishables Handling Quaterly. 396

95, 5-6. 397

398

Schroeder, J. I., Allen, G. J., Hugouvieux, V., Kwak, J. M. & Waner, D. (2001). Guard 399

Cell Signal Transduction. Annual Review of Plant Physiology and Plant Molecular 400

Biology, 52, 627-658. doi: 10.1146/annurev.arplant.52.1.627. 401

402

She, X.P. & Song, X.G. (2012). Ethylene inhibits abscisic acid-induced stomatal closure 403

in Vicia faba via reducing nitric oxide levels in guard cells. New Zealand Journal of 404

Botany, 50, 203-216. https://doi.org/10.1080/0028825X.2012.661064. 405

406

Tanaka, Y., Sano, T., Tamaoki,M., Nakajima, N., Kondo, N. & Hasezawa, S. (2015). 407

Ethylene Inhibits Abscisic Acid-Induced Stomatal Closure in Arabidopsis. Plant 408

24

Physiology, 138, 2337–2343. https://doi.org/10.1104/pp.105.063503. 409

410

Telewski, F. W. & Jaffe, M. J. (1986). Thigmomorphogenesis: The role of ethylene in the 411

response of Pinus taeda and Abies fraseri to mechanical perturbation. Physiologia 412

Plantarum, 66, 227-233. https://doi.org/10.1111/j.1399-3054.1986.tb02413.x 413

414

Vishal, B. & Kumar, P. P. (2018). Regulation of Seed Germination and Abiotic Stresses 415

by Gibberellins and Abscisic Acid. Frontiers in Plant Science, 9, 838. 416

https://doi.org/10.3389/fpls.2018.00838. 417

418

Wang, J., Ma H. & Wang, S. (2019). Application of Ultrasound, Microwaves, and 419

Magnetic Fields Techniques in the Germination of Cereals. Food Science and Technology 420

Research, 25, 489-497. https://doi.org/10.3136/fstr.25.489. 421

422

Yang, S. F. (2003). Ethylene Biosynthesis and its Regulation in Higher Plants. Annual 423

Review of Plant Physiology, 35, 155-189. DOI: 10.1146/annurev.pp.35.060184.001103. 424

425

Yang, H., Gao, J., Yang, A. & Chen, H. (2015). The ultrasound-treated soybean seeds 426

25

improve edibility and nutritional quality of soybean sprouts. Food Research International, 427

77, 704-710. https://doi.org/10.1016/j.foodres.2015.01.011. 428

429

Yang, Y., Wu, Y., Pirrello, J., Regad, F., Bouzayen, M., Deng, W. & Li, Z. (2010). 430

Silencing Sl-EBF1 and Sl-EBF2 expression causes constitutive ethylene response 431

phenotype, accelerated plant senescence, and fruit ripening in tomato. Journal of 432

Experimental Botany, 61, 679-708. https://doi.org/10.1093/jxb/erp332. 433

434

Yang, S. F. & Hoffman, N. E. (1984). Ethylene biosynthesis and its regulation in higher 435

plants. Annual Review of Plant Physiology, 35, 155–189. 436

https://doi.org/10.1146/annurev.pp.35.060184.001103. 437

438

Yu, J., Engeseth, N. J. and Feng, H. (2016). High Intensity Ultrasound as an Abiotic 439

Elicitor—Effects on Antioxidant Capacity and Overall Quality of Romaine Lettuce. Food 440

and Bioprocess Technology, 9, 262–273. DOI: 10.1007/s11947-015-1616-7. 441

442

Zhou, B., Feng, H. & Pearlstein, A.J. (2012). Continuous-flow ultrasonic washing system 443

for fresh produce surface decontamination. Innovative Food Science & Emerging 444

26

Technologies, 16, 427-435. https://doi.org/10.1016/j.ifset.2012.09.007. 445

446

Zhu J.K. (2002). Salt and drought stress signal transduction in plants. Annual Review of 447

Plant Biology, 53, 247-273. doi: 10.1146/annurev.arplant.53.091401.143329. 448

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

https://www.editorialmanager.com/fochms/download.aspx?id=3303&guid=af67be84-ca83-474a-b7ef-381cf4103e47&scheme=1https://www.editorialmanager.com/fochms/download.aspx?id=3303&guid=af67be84-ca83-474a-b7ef-381cf4103e47&scheme=1

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.


https://www.editorialmanager.com/fochms/download.aspx?id=3304&guid=880c8cd0-8e4b-4642-bba3-cf0f0eca7863&scheme=1https://www.editorialmanager.com/fochms/download.aspx?id=3304&guid=880c8cd0-8e4b-4642-bba3-cf0f0eca7863&scheme=1

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


https://www.editorialmanager.com/fochms/download.aspx?id=3305&guid=7a4ff311-a3e0-429f-b041-2915db54b50d&scheme=1https://www.editorialmanager.com/fochms/download.aspx?id=3305&guid=7a4ff311-a3e0-429f-b041-2915db54b50d&scheme=1

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.


https://www.editorialmanager.com/fochms/download.aspx?id=3306&guid=ef2e38a9-6b38-4206-88ba-2f1131786e6b&scheme=1https://www.editorialmanager.com/fochms/download.aspx?id=3306&guid=ef2e38a9-6b38-4206-88ba-2f1131786e6b&scheme=1

GraphicA

bstract_2.ai ��

Graphical abstract Click here to access/download;Figure;GraphicAbstract.tif

https://www.editorialmanager.com/fochms/download.aspx?id=3285&guid=f2af38d0-30b6-4464-870e-cf04c8ace089&scheme=1https://www.editorialmanager.com/fochms/download.aspx?id=3285&guid=f2af38d0-30b6-4464-870e-cf04c8ace089&scheme=1

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.

Table1 Click here to access/download;Table;Table 1.xlsx

https://www.editorialmanager.com/fochms/download.aspx?id=3302&guid=a5d25121-be36-4273-8b03-bfa574d57ea5&scheme=1https://www.editorialmanager.com/fochms/download.aspx?id=3302&guid=a5d25121-be36-4273-8b03-bfa574d57ea5&scheme=1

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

Click here to access/downloadSupplementary Material

Supplementary Table 1.xlsx

https://www.editorialmanager.com/fochms/download.aspx?id=3308&guid=2cdda05a-6641-49b6-a342-8650142869fc&scheme=1




https://www.editorialmanager.com/fochms/download.aspx?id=3309&guid=4a5f1aef-24a4-471c-b9cd-d7be40d2f537&scheme=1




https://www.editorialmanager.com/fochms/download.aspx?id=3310&guid=1325cc69-5231-48c2-bba2-326c2247fdf2&scheme=1




https://www.editorialmanager.com/fochms/download.aspx?id=3311&guid=2b6ff7e4-1574-48ca-8bc8-b2da1ab27944&scheme=1



Supplementary table 5.xlsx

https://www.editorialmanager.com/fochms/download.aspx?id=3312&guid=ad7111a4-0429-4018-9997-0f8caa28c879&scheme=1



Supplementary table 6.xlsx

https://www.editorialmanager.com/fochms/download.aspx?id=3313&guid=ef5b7add-b9df-4a72-9b9f-51f256cc7208&scheme=1




https://www.editorialmanager.com/fochms/download.aspx?id=3314&guid=631163ec-f910-4c30-b3a1-b1fa98db338d&scheme=1




https://www.editorialmanager.com/fochms/download.aspx?id=3315&guid=779e5bac-713f-4d39-ab29-d8694c5b68bd&scheme=1




https://www.editorialmanager.com/fochms/download.aspx?id=3316&guid=4d32fa62-5439-4c93-a04c-75a6d306f0f3&scheme=1



https://www.editorialmanager.com/fochms/download.aspx?id=3317&guid=80c5dd40-ff52-48e5-bec0-e05128c9c02f&scheme=1

food chemistry: molecular sciences...cabbage, lettuce) was retained for a long time after ultrasonic...

Documents