Running title: Low temperature promotes peel degreening in citrus fruit … · Running title: Low temperature promotes peel degreening in citrus fruit Low temperature transcriptionally

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Running title: Low temperature promotes peel degreening in citrus fruit 1

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Low temperature transcriptionally modulates natural peel degreening in lemon (Citrus 4

limon L.) fruit independently of endogenous ethylene 5

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Oscar W. Mitalo1, Takumi Otsuki1, Rui Okada1, Saeka Obitsu1, Kanae Masuda1, Yuko Hojo2, 7

Takakazu Matsuura2, Izumi C. Mori2, Daigo Abe3, William O. Asiche4, Takashi Akagi1, 8

Yasutaka Kubo1*, Koichiro Ushijima1* 9

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1 Graduate School of Environmental and Life Science, Okayama University, Okayama, 700–11

8530, Japan 12

2 Institute of Plant Science and Resources, Okayama University, Kurashiki, 700–0046, Japan 13

3 National Agriculture and Food Research Organization, Shikoku Research Station, Zentsuji, 14

765–8508, Japan 15

4 Department of Research and Development, Del Monte Kenya Ltd, Thika, 147–01000, 16

Kenya 17

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Email addresses of authors: Oscar W. Mitalo ([email protected]), Takumi Otsuki 19

([email protected]), Rui Okada ([email protected]), Saeka Obitsu 20

([email protected]), Kanae Masuda ([email protected]), Yuko 21

Hojo ([email protected]), Takakazu Matsuura ([email protected]), 22

Izumi C. Mori ([email protected]), Daigo Abe ([email protected]), William O. Asiche 23

([email protected]), Takashi Akagi ([email protected]) 24

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* Corresponding authors: Yasutaka Kubo (Email: [email protected], Tel.: +81-86-26

251-8338); Koichiro Ushijima (Email: [email protected], Tel.: +81-86-251-27

8355) 28

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Date of submission: 26 November 2019 30

Number of figures: 8; Colour in print: 8 31

Word count: 6271 32

Supplementary figures: 1 33

Supplementary tables: 11 34

.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted November 27, 2019. . https://doi.org/10.1101/855775doi: bioRxiv preprint








https://doi.org/10.1101/855775

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Highlight: 35

Citrus peel degreening is promoted by low temperature via modulation of multiple genes 36

associated with chlorophyll degradation, carotenoid biosynthesis, photosystem disassembly, 37

phytohormones and transcription factors without involving ethylene signalling. 38

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Abstract 40

Peel degreening is an important aspect of fruit ripening in many citrus fruit, and earlier 41

studies have shown that it can be advanced either by ethylene treatment or during low 42

temperature storage. However, the important regulators and pathways involved in natural 43

peel degreening remain largely unknown. To understand how natural peel degreening is 44

regulated in lemon (Citrus limon L.) fruit, flavedo transcriptome and physiochemical changes 45

in response to either ethylene treatment or low temperature were studied. Ethylene treatment 46

induced rapid peel degreening which was strongly inhibited by the ethylene antagonist, 1-47

methylcyclopropene (1-MCP). Compared with 25ºC, moderately low temperatures (5ºC, 48

10ºC, 15ºC and 20ºC) also triggered peel degreening. Surprisingly, repeated 1-MCP 49

treatments failed to inhibit the peel degreening induced by low temperature. Transcriptome 50

analysis revealed that low temperature and ethylene independently regulated genes associated 51

with chlorophyll degradation, carotenoid metabolism, photosystem proteins, phytohormone 52

biosynthesis and signalling, and transcription factors. On-tree peel degreening occurred along 53

with environmental temperature drops, and it coincided with the differential expression of 54

low temperature-regulated genes. In contrast, genes that were uniquely regulated by ethylene 55

showed no significant expression changes during on-tree peel degreening. Based on these 56

findings, we hypothesize that low temperature plays a prominent role in regulating natural 57

peel degreening independently of ethylene in citrus fruit. 58

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Keywords: 1-methylcyclopropene, Carotenoids, Chlorophyll, Citrus, Ethylene, Low 60

temperature, Peel degreening, Phytohormones, Transcriptome 61


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Introduction 62

Fruit ripening is a multifaceted process comprising various physiochemical and structural 63

changes such as softening, starch degradation to sugars, colour development and aroma 64

volatile production (Cherian et al., 2014; Seymour and Granell, 2014). In citrus fruit, colour 65

development, commonly known as peel degreening, is a critical part of fruit ripening which is 66

characterized by peel colour change from green to yellow/red/orange (Iglesias et al., 2007). 67

Peel degreening is an important aspect for marketability of citrus fruit (Porat, 2008), and thus 68

there is wide interest in unravelling the fundamental regulatory mechanisms involved. 69

70

There are two main pathways that have been linked to citrus peel degreening. One is 71

chlorophyll degradation, which firstly involves dephytilation of chlorophyll molecules by 72

chlorophyllase (CLH) and pheophytinase (PPH) followed by removal of the central Mg atom 73

by Mg-dechelatase to form pheophorbide. Pheophorbide is then converted to red chlorophyll 74

catabolites (RCC) by pheophorbide a oxidase (PaO), and RCC is reduced to colourless 75

compounds by RCC reductase (RCCR) (Hörtensteiner, 2006; Shimoda et al., 2016; Yin et al., 76

2016). The other is carotenoid biosynthesis which starts with the condensation of two 77

geranylgeranyl pyrophosphate (GGPP) molecules by phytoene synthase (PSY) to form 78

phytoene. Phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) successively convert 79

phytoene to lycopene, which is then converted to either α-carotene or β-carotene by lycopene 80

ε-cyclase (LCYe) and lycopene β-cyclase (LCYb) respectively. α-carotene is later converted 81

to lutein via sequential hydroxylation by ε-ring hydroxylase and β-ring hydroxylase (CHYb), 82

whereas β-carotene is converted to zeaxanthin via β-cryptoxanthin by CHYb (Cunningham et 83

al., 1996; Ohmiya et al., 2019). Genes encoding various enzymes for the main steps of 84

chlorophyll degradation and carotenoid metabolism have been isolated and functionally 85

characterized (Rodrigo et al., 2013). 86

87

The phytohormone ethylene has been routinely used for commercial degreening in citrus fruit 88

(Purvis and Barmore, 1981; Porat, 2008; Mayuoni et al., 2011). Exogenous ethylene 89

application was shown to transcriptionally modulate both chlorophyll degradation and 90

carotenoid metabolism (Rodrigo and Zacarias, 2007; Shemer et al., 2008; Yin et al., 2016). 91

Transcription factors (TF) that may be involved in ethylene-induced peel degreening have 92

also been identified and characterized (Yin et al., 2016). Nevertheless, it remains unclear 93

whether ethylene plays a role during natural degreening since citrus fruit are non-climacteric 94


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and the amounts of ethylene produced are minute (Eaks, 1970; Sawamura, 1981; Katz et al., 95

2004). 96

97

Temperature has a large impact on a wide range of plant growth and developmental processes, 98

including fruit ripening and maturation. Low temperature is thought to slow most cell 99

metabolic activities, and hence it is the major postharvest technology used to delay fruit 100

ripening and senescence (McGlasson et al., 1979; Hardenburg et al., 1986). However, 101

promotion of fruit ripening by low temperature has been described in various fruit species 102

such as kiwifruit (Kim et al., 1999; Mworia et al., 2012; Asiche et al., 2017; Mitalo et al., 103

2018a), European pears (El-Sharkawy et al., 2003; Nham et al., 2017), and apples (Tacken et 104

al., 2010). Recently, transcriptome studies have suggested that low temperature-specific 105

genes might have regulatory roles during fruit ripening in kiwifruit (Asiche et al., 2018; 106

Mitalo et al., 2018b; Mitalo et al., 2019a; Mitalo et al., 2019b) and European pears (Mitalo et 107

al., 2019c). 108

109

Citrus fruit are among the species where low temperature has been linked to fruit ripening, 110

especially peel degreening. Typically, as most citrus fruit mature on the tree, the seasonal 111

temperature drops. Multiple studies have thus demonstrated that cold periods below 13ºC are 112

required to stimulate on-tree fruit colour development (Manera et al., 2012; Manera et al., 113

2013; Rodrigo et al., 2013; Conesa et al., 2019). During storage, low/intermediate 114

temperatures (6–15ºC) have also been shown to promote peel degreening (Matsumoto et al., 115

2009; Van Wyk et al., 2009; Zhu et al., 2011; Carmona et al., 2012a; Tao et al., 2012). 116

Natural peel degreening in citrus fruit has often been attributed to ethylene, on the 117

assumption that the basal system I ethylene levels are physiologically active (Goldschmidt et 118

al., 1993; Carmona et al., 2012b). Whether the peel colour changes during on-tree maturation 119

and low temperature storage are caused by basal ethylene, low temperature and/or a 120

synergistic effect of ethylene and low temperature, or because of another mechanism is still 121

not yet clear. 122

123

Here, we examined the peel degreening behaviour of lemon fruit in response to exogenous 124

ethylene and different storage temperatures (0ºC, 5ºC, 10ºC, 15ºC, 20ºC, and 25ºC). We 125

found that both ethylene treatment and moderately low temperatures (0ºC, 5ºC, 10ºC, 15ºC 126

and 20ºC; hereinafter referred to as low temperature) promoted peel degreening. Further, we 127

explored the role of ethylene in low temperature-triggered peel degreening using repeated 128


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treatments with 1-methylcyclopropene (1-MCP), a well-known ethylene antagonist (Watkins, 129

2006; Sisler and Serek, 1997). We found that 1-MCP treatments did not inhibit the 130

accelerated peel colour changes induced by low temperature. Further transcriptome analysis 131

revealed that ethylene and low temperature independently regulated distinct gene sets in the 132

flavedo of lemon fruit. On-tree peel degreening also coincided with a decrease in minimum 133

environmental temperatures and differential expression of low temperature-regulated genes, 134

whereas ethylene-specific genes showed no significant expression changes. These results 135

suggested that low temperature might transcriptionally modulate peel degreening 136

independently of basal endogenous ethylene. 137

138

Materials and methods 139

Plant material and treatments 140

Lemon fruit (C. limon L. cv. ‘Allen Eureka’) grown under standard cultural practices were 141

collected in 2018 from a commercial orchard in Takamatsu (Kagawa, Japan). Sampling was 142

from seven harvests during fruit development: 3 Sep., 27 Sep., 12 Oct., 30 Oct., 14 Nov., 29 143

Nov., and 13 Dec., corresponding to 171, 196, 211, 230, 246, 261 and 276 days after full 144

bloom (DAFB), respectively. To characterize postharvest ethylene effect, lemons (196 145

DAFB) were divided into four lots of 20 fruit each. The first set of fruit contained non-treated 146

fruit (control), while the second set were treated with 1-MCP (2 µLL–1) for 12 h. The third set 147

of fruit were continuously treated with ethylene (100 µLL–1), while the fourth set were 148

initially treated with 1-MCP (2 µLL–1) for 12 h followed by continuous ethylene (100 µLL–1) 149

treatment. 1-MCP was released by dissolving SmartFresh™ powder (AgroFresh, PA, United 150

States) in water. All treatments were carried out at 25ºC for up to 8 d. For postharvest storage 151

tests, lemons (196 DAFB) were divided into five lots of 40 fruit each, and stored at either 5ºC, 152

10ºC, 15ºC, 20ºC or 25ºC for up to 42 d. Additionally, three separate sets (40 fruit each) were 153

stored at either 5ºC, 15ºC or 25ºC with repeated (twice a week) 12 h 1-MCP treatments. Fruit 154

peel (flavedo) was sampled, frozen in liquid nitrogen and stored at -80ºC for future analysis, 155

each sample containing three biological replicates. 156

157

Citrus colour index (CCI) determination 158

The L, a and b Hunter lab parameters were measured on four even equatorial sites on the fruit 159

surface using a Minolta CR-200B chromameter (Konica Minolta, Tokyo, Japan). CCI values 160

were presented as the results of 1000•a/L•b transformation, expressed as a mean of five fruit. 161


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162

Determination of chlorophyll and carotenoid content 163

Chlorophylls were extracted and quantified in triplicate according to the procedure by 164

Rodrigo et al. (2003) with slight modifications. Chlorophylls were extracted in 80% acetone 165

and appropriate dilutions were used to quantify absorbance at 646.8 nm and 663.2 nm. 166

Chlorophyll content was calculated from these measurements using Lichtenthaler and 167

Wellburn equations (Wellburn, 1994). Extraction and quantification of carotenoids were 168

conducted in triplicate according to the procedures by Kato et al. (2004) and Matsumoto et al. 169

(2007) with slight modifications. Briefly, carotenoids were successively extracted with 40% 170

methanol and diethyl ether/methanol (containing 0.1% BHT). After saponification with 171

methanolic potassium hydroxide, the organic layer of the extracts was vacuum-dried and 172

analysed by HPLC. The HPLC analysis was carried out on an Extrema LC-4000 system 173

(Jasco, Tokyo, Japan) equipped with a photo diode-array detector and autosampler. Samples 174

were analysed on a Develosil C30-UG column (3 µm, 150 x 4.6 mm, Nomura Chemicals, 175

Aichi, Japan) set at 20ºC and 0.5 mL/min flow rate. The UV-Vis spectra were obtained 176

between 250 and 550 nm, and chromatograms were processed at 450 nm. Carotenoid 177

quantifications were based on standard curves generated using authentic standards. 178

179

Phytohormone measurements 180

Phytohormone extraction and analysis were performed according to the method described by 181

Gupta et al. (2017), using deuterium-labelled internal standards for indole-3-acetic acid 182

(IAA), abscisic acid (ABA), jasmonic acid (JA), gibberellins (GAs), trans-zeatin (tZ), N6-183

isopentenyladenine (iP) and salicylic acid (SA), and 13C-labelled jasmonoyl-L-isoleucine 184

(JA-Ile). Eluted fractions were analysed on an Agilent 1260-6410 Triple Quad LC/MS 185

system equipped with a ZOR-BAX Eclipse XDB-C18 column (Agilent Technologies, CA, 186

USA). Liquid chromatography conditions are described in Table S1, while the multiple-187

reaction-monitoring mode of the tandem quadrupole mass spectrometer and precursor-188

product ion transitions for each compound are listed in Table S2. 189

190

RNA-seq and differential gene expression analysis 191

Total RNA was extracted in triplicate from the flavedo of ethylene-treated and control (non-192

treated) lemon fruit after 4 d, as well as fruit after 28 d of storage at 5ºC, 15ºC and 25ºC. 193

Illumina paired-end libraries were constructed using NEBNext® Ultra™ RNA Library Prep 194


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Kit for Illumina (New England Biolabs, MA, USA), before being sequenced using Illumina 195

HiSeq 2500 platform (Hokkaido System Co. Ltd., Japan). Trimming was done to obtain ≥ 10 196

million paired reads per sample, and the reads were mapped to the reference Citrus 197

clementina Genome v1.0 (Wu et al., 2014). Gene expression levels were calculated using the 198

reads by kilobase per million (RPKM) method and differentially expressed genes (DEGs) 199

were identified using the false discovery rates (FDR) analysis (Robinson et al., 2010). DEG 200

selection was based on two criteria: (i) genes with RPKM ≥ 3.0 and FDR ≤ 0.001, and (ii) 201

fold change ≥ 3.0 in average RPKM for ethylene vs. control, 5ºC vs. 25ºC and/or 15ºC vs. 202

25ºC. For co-expression analysis, the WGCNA method (Zhang and Horvath, 2005) was used 203

to generate modules of highly correlated genes based on the RNA-seq expression data. Gene 204

modules were identified by implementing the WGCNA package in R (Langfelder and 205

Horvath, 2008). The soft-thresholding power and tree-cut parameters used for the WGCNA 206

analysis were 12 and 0.15, respectively. 207

208

Reverse-transcriptase quantitative PCR (RT-qPCR) 209

Total RNA was extracted from the flavedo of fruit at harvest (0 d), after 4 d for ethylene, 1-210

MCP + ethylene, and control groups, and after 28 d storage at 5ºC, 10ºC, 15ºC, 20ºC and 211

25ºC. Total RNA was also extracted from on-tree fruit samples at each of the specified 212

sampling dates. DNase I (Nippon Gene, Tokyo, Japan) treatment followed by clean-up using 213

FavorPrep after Tri-Reagent RNA Clean-up Kit (Favorgen Biotech. Co., Ping-Tung, Taiwan) 214

were carried out to remove genomic DNA contamination from the extracted RNA. For all 215

treatments, 2.4 µg of clean RNA was reverse-transcribed to cDNA using Takara RNA PCR 216

kit (Takara, Kyoto, Japan). Gene-specific primers (Table S3) were designed using Primer3 217

software (version 0.4.0, http://bioinfo.ut.ee/primer3-0.4.0/). Gene expression of three 218

biological replicates was examined on MYiQ Single-Color Reverse Transcriptase-219

Quantitative PCR Detection System (Bio-Rad, Hercules, CA, USA) using TB Green™ 220

Premix ExTaq™ II (Takara, Kyoto, Japan). AcActin (Ciclev10025866m.g) was used as the 221

housekeeping gene after examining its constitutive expression pattern from the RNA-seq 222

results. Relative expression values were calculated using 196 DAFB (0 d) fruit. 223

224

Statistical analysis 225

Data presented in this study were subjected to statistical analysis using R version 3.4.0 226

software package (R Project). ANOVA followed by post-hoc Tukey’s test (P < 0.05) were 227


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used to detect statistical differences in CCI, pigment and phytohormone contents, and gene 228

expression. 229

230

Results 231

Ethylene-induced peel degreening 232

To validate the role of ethylene in citrus peel degreening, detached lemon fruit were 233

continuously treated with ethylene and/or its antagonist, 1-MCP. As expected, peel colour of 234

ethylene-treated fruit started to change from green to yellow after 2 d, attaining a full yellow 235

colour after 8 d (Fig. 1A). This change was numerically indicated by a rapid increase in CCI 236

from -14.2 at 0 d to -1.8 after 8 d. Notably, fruit pre-treated with 1-MCP followed by 237

continuous ethylene treatment retained their greenish peel colour and CCI showed no 238

significant changes throughout the experimental period. These findings demonstrated that 1-239

MCP pre-treatment rendered the fruit insensitive to ethylene, effectively inhibiting ethylene 240

action on the peel colour. 241

242

Peel degreening behaviour at different storage temperatures and the effect of 1-MCP 243

Peel colour changes in detached lemon fruit were also investigated during storage at different 244

temperatures. As shown in Fig. 1B, peel colour of fruit at 5ºC, 10ºC, 15ºC and 20ºC gradually 245

changed from green to yellow with a concomitant increase in CCI to about -2.3 after 28–42 d. 246

Peel degreening was more pronounced at 15ºC followed by 10ºC and 20ºC, than 5ºC at which 247

fruit retained appreciable greenish colour even after 42 d. In contrast, fruit at 25ºC retained 248

their greenish peel colour and the CCI changes were minimal throughout the storage period. 249

These observations indicated that moderately low storage temperatures promoted the peel 250

degreening process in lemon fruit. 251

252

To determine whether the basal levels of system I ethylene played a role in the observed low 253

temperature-modulated peel degreening, we treated lemon fruit repeatedly with 1-MCP. 254

Surprisingly, comparable peel degreening was observed in fruit at 5ºC and 15ºC but not at 255

25ºC, notwithstanding the repeated 1-MCP treatments (Fig. 1C). Together, these findings 256

suggested that low temperature may promote peel colour changes in lemon fruit 257

independently of ethylene. 258

259

Differential expression analysis in lemon fruit flavedo 260

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Overview of the transcriptome changes 262

To gain a deeper insight into the mechanisms of low temperature promotion of peel 263

degreening, we conducted a comprehensive transcriptome analysis to compare low 264

temperature-induced responses with those activated by ethylene. Ethylene-induced responses 265

were captured by examining 4 d ethylene-treated and non-treated (control) flavedo samples. 266

To cover low temperature-triggered responses, samples obtained after 28 d of storage at 5ºC 267

and 15ºC were examined against those at 25ºC. 268

269

RNA-seq analysis resulted in the identification of 3105 DEGs (q-value < 0.001), which 270

responded to either ethylene or low temperature (Fig. 2). Ethylene had the largest share, 271

influencing 2329 DEGs as opposed to 5ºC and 15ºC that influenced 1634 and 597 DEGs, 272

respectively (Fig. 2A). In all treatments, the number of downregulated DEGs was higher than 273

that of upregulated genes. Clustering analysis classified the DEGs into distinct groups that 274

were regulated by either ethylene, 5ºC and/or 15ºC (Fig. 2B). Ethylene treatment exclusively 275

upregulated and downregulated 592 and 700 genes, respectively (Fig. 2C, D). Likewise, an 276

aggregate of 337 and 439 genes were exclusively upregulated and downregulated, 277

respectively by 5ºC and 15ºC. The remaining DEGs (420 upregulated and 617 278

downregulated) were jointly influenced by either ethylene, 5ºC and/or 15ºC. Detailed 279

information about the DEGs showing specific and shared responses to ethylene, 5ºC and/or 280

15ºC is listed in Tables S4–S10. Altogether, identified DEGs could be pooled into three 281

distinct groups. The first group comprised ethylene-specific genes, the second group included 282

low temperature-specific genes while the third group consisted of genes regulated by either 283

ethylene or low temperature. 284

285

Chlorophyll metabolism and associated transcripts 286

Peel degreening is primarily caused by the degradation of green-coloured chlorophyll 287

pigments to colourless non-fluorescent derivatives (Hortensteiner, 2006). Upon ethylene 288

treatment for 4 d, peel chlorophyll a and b content drastically decreased from about 50 µg g–1 289

at harvest to merely 11 µg g–1 (Fig. 3A). However, ethylene treatment failed to induce 290

chlorophyll reduction in fruit pre-treated with 1-MCP which was in close agreement with the 291

observed colour changes (Fig. 1A). During storage, peel chlorophyll content also decreased 292

in fruit at moderately low temperatures (5ºC, 10ºC, 15ºC and 20ºC), whereas they were 293

maintained at high levels in fruit at 25ºC. It is noteworthy that peel chlorophyll content also 294

decreased in fruit at 5ºC and 15ºC despite repeated treatments with 1-MCP. 295


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The acceleration of chlorophyll loss by ethylene and low temperature was further verified by 297

examining the expression of chlorophyll metabolism genes. Whereas most of the identified 298

DEGs encoding chlorophyll metabolism enzymes were downregulated, we found three that 299

were upregulated (Fig. 3B, Table S11). Among the upregulated genes were ClCLH1 and 300

ClPPH, that had been previously associated with chlorophyll degradation in citrus fruit 301

(Jacob-Wilk et al., 1999; Yin et al., 2016). Interestingly, ClCLH1 was up-regulated only by 302

ethylene treatment (which was suppressed by 1-MCP treatment), while ClPPH was 303

upregulated by both ethylene treatment and low temperature (Fig. 3C). It is however worth 304

noting that repeated 1-MCP treatments did not suppress the increased expression of ClPPH at 305

5ºC and 15ºC. 306

307

Carotenoid metabolism and associated transcripts 308

Citrus peel degreening is also complemented by a change in the content and composition of 309

carotenoids having varied colours (Kato, 2012; Ohmiya et al., 2019). Therefore, we sought to 310

determine the changes in peel carotenoid content triggered by ethylene treatment and storage 311

temperature. Lutein, β-carotene and α-carotene were identified as the major carotenoids in the 312

peel of lemon fruit (Fig. S1), which was in close agreement with the findings of Agócs et al. 313

(2007). Interestingly, the peel content of all the identified carotenoids showed a substantial 314

decrease upon ethylene treatment for 4 d and storage at moderately low temperatures for 28 d 315

(Fig. 4A). However, while 1-MCP treatment significantly inhibited carotenoid changes 316

induced by ethylene treatment, repeated 1-MCP treatments did not abolish peel carotenoid 317

decrease at 5ºC and 15ºC. 318

319

By examining the RNA-seq data, we identified 13 DEGs that had been associated with 320

carotenoid metabolism (Fig. 4B, Table S11). Out of these, three genes including ClPSY1, 321

ClLCYb2a and ClCHYb1 that showed high RPKM values and unique expression patterns 322

were selected for further analysis by RT-qPCR. This analysis revealed that ClPSY1 and 323

ClLCYb2a were upregulated by both ethylene treatment and low temperature, while 324

ClCHYb1 was upregulated exclusively by low temperature (Fig. 4C). Additionally, the 325

expression of all the three analysed genes increased in the peel of fruit at 5ºC and 15ºC 326

despite the repeated 1-MCP treatments. 327

328

Transcripts encoding photosystem proteins 329


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Genes encoding photosystem proteins featured prominently among the identified DEGs, and 330

most of them were downregulated by both ethylene treatment and low temperature (Fig. 5A, 331

Table S11). However, ethylene treatment appeared to have a greater influence on their 332

downregulation than low temperature did. Since most of genes in this category showed a 333

similar expression pattern, we selected only one, light harvesting complex 2 (ClLHCB2) for 334

validation and further analysis by RT-qPCR. Results confirmed that both ethylene treatment 335

and low temperature caused a reduction in the expression of ClLHCB2 (Fig. 5B). 336

Nevertheless, repeated 1-MCP treatments did not suppress the expression decrease induced at 337

5ºC and 15ºC, suggesting that the influence of low temperature on ClLHCB2 expression was 338

independent of ethylene. 339

340

Phytohormone levels and associated transcripts 341

Another prominent category among the identified DEGs included genes that were associated 342

with the biosynthesis and signalling of phytohormones, especially ethylene, JA, ABA, auxin 343

and GA (Fig. 6A). Most of the ethylene-related genes were up-regulated by ethylene 344

treatment, while low temperature, especially 5ºC, only showed a slight effect on their 345

expression. On the other hand, genes that were associated with JA and ABA were mostly 346

upregulated by both ethylene treatment and low temperature. Auxin-related genes showed 347

varied expression patterns, although the general trend was towards a downregulation by both 348

ethylene treatment and low temperature. We also identified three GA-associated DEGs of 349

which one (ClGA20ox2), which is associated with GA biosynthesis, was downregulated by 350

both ethylene treatment and low temperature, especially at 5ºC. In contrast, ClGA2ox4 and 351

ClGA2ox8 that are associated with GA degradation were upregulated by ethylene treatment 352

as well as low temperature. To verify the roles of ethylene and low temperature in the 353

regulation of phytohormone-related genes, 9-cis-epoxycarotenoid dioxygenase (ClNCED5) 354

which is associated with ABA biosynthesis was chosen for further analysis by RT-qPCR. 355

Results confirmed that ClNCED5 was up-regulated both after 4 d of ethylene exposure, and 356

28 d of storage at lower temperatures (5ºC, 10ºC, 15ºC and 20ºC) than 25ºC (Fig. 6B). There 357

was also a significant increase in ClNCED5 expression in fruit that were repeatedly treated 358

with 1-MCP at 5ºC and 15ºC. The transcript levels of ClNCED5 were notably higher in low 359

temperature-stored fruit than in ethylene-treated fruit. 360

361

The above changes in expression of phytohormone-associated genes motivated us to 362

determine the phytohormone content in the flavedo of lemon fruit exposed to ethylene and 363


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different storage temperatures. The results indicated that both ethylene treatment and low 364

storage temperature caused a significant hike in ABA and JA-Ile levels (Fig. 6C). In 365

particular, both ABA and JA-Ile levels were substantially higher in fruit stored at low 366

temperatures than in ethylene-treated fruit. Unfortunately, we could not detect the other 367

hormones because of their extremely low endogenous levels and severe ion suppression 368

effects during LC/MS analysis. 369

370

Transcripts encoding transcription factors 371

A total of 128 DEGs in the RNA-seq data were found to encode a wide range of putative TF 372

families including AP2/ERF, bHLH, MYB, NAC, GRAS, zinc finger, homeobox, WRKY, 373

MADS and TCP (Fig. 7A, Table S11). This finding underscored the relevance of TF activity 374

in the peel degreening process of lemon fruit. Identified genes were therefore pooled into 375

three distinct groups, which included those that were influenced by (i) ethylene only such as 376

ClERF114, (ii) low temperature only such as ClERF3, and (iii) both ethylene and low 377

temperature such as ClbHLH25. RT-qPCR analysis confirmed that ClERF114 was 378

exclusively upregulated by ethylene treatment as its expression was maintained at minimal 379

levels during storage (Fig. 7B). In contrast, ClERF3 was exclusively upregulated by low 380

temperature since marginal expression levels were registered in ethylene-treated fruit (Fig. 381

7C). Finally, ClbHLH25 expression increased both upon ethylene treatment and after storage 382

at lower temperatures than 25ºC (Fig. 7C). It is also noteworthy that repeated 1-MCP 383

treatments failed to abolish the upregulation of ClERF3 and ClbHLH25 at 5ºC and 15ºC (Fig. 384

7B, C). 385

386

On-tree peel degreening behaviour and expression analysis of associated genes 387

The roles of ethylene and low temperature in natural peel degreening were further 388

investigated during on-tree maturation of lemon fruit. For this purpose, fruit were harvested 389

at seven progressive stages ranging from 176 to 276 DAFB that occurred between early-390

September and mid-December. As shown in Fig. 8A, peel colour progressively changed 391

from green on 3rd September to full yellow on 13th December, which was indicated by a 392

concomitant increase in CCI from -16.3 to -1.1 within the same time span. As peel 393

degreening progressed, the average minimum temperatures in the orchard location decreased 394

gradually from 22.5ºC on 3rd September to 3.7ºC on 13th December. The increase in CCI was 395

initially slow between 3rd September to 12th October from -16.3 to -14.2 when the minimum 396

temperatures were above 13ºC. Interestingly, CCI increased rapidly from -14.2 to -1.1 397


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between 12th October and 13th December when the minimum temperatures were maintained 398

at below 13ºC. The observed loss of green colour during on-tree maturation was in close 399

agreement with a gradual decrease in the peel chlorophyll a and b contents (Fig. 8B). Equally, 400

peel degreening was also accompanied by a gradual decline in the peel content of lutein, β-401

carotene and α-carotene (Fig. 8C). 402

403

The correlation between peel colour changes and environmental temperature drops was 404

further investigated by examining the expression patterns of selected genes induced by 405

ethylene and/or low temperature from the RNA-seq data. On-tree peel degreening coincided 406

with an upregulation of ClPPH and downregulation of ClLHCB2 (Fig. 8D), both of which 407

were earlier shown to be influenced by low temperature (Fig. 3C, 5B). However, the 408

ethylene-specific ClCLH1 did not show any significant changes in expression. On-tree peel 409

degreening was also accompanied by the upregulation of all the three analysed carotenoid 410

metabolism genes ClPSY1, ClLCYb2a and ClCHYb1 (Fig. 8E), which were earlier shown to 411

be upregulated by low temperature (Fig. 4C). Among the TF-encoding genes, the ethylene-412

specific ClERF114 did not show any significant expression changes, whereas both ClERF3 413

and ClbHLH25 were upregulated especially from 30th October when the minimum 414

temperatures were below 13ºC (Fig. 8F). Altogether, these observations demonstrated strong 415

similarities between on-tree and low temperature-modulated peel degreening, as well as their 416

dissimilarities with ethylene-induced changes. 417

418

Discussion 419

Many studies have shown that ethylene regulates peel degreening in citrus fruit (Purvis and 420

Barmore, 1981; Shemer et al., 2008; Yin et al., 2016), prompting its wide use for commercial 421

degreening purposes (Porat, 2008; Mayuoni et al., 2011). This is consistent with the present 422

study as ethylene treatment induced rapid peel degreening in detached lemon fruit (Fig. 1A). 423

However, the important regulators involved in natural peel degreening remain a mystery 424

since citrus fruit are considered non-climacteric, and thus produce trace levels of endogenous 425

ethylene (system I ethylene) (Katz et al., 2004). Previous studies have demonstrated that 426

there is a close association between low temperature and peel colouration in multiple citrus 427

fruit species (Carmona et al., 2012a; Manera et al., 2012; Manera et al., 2013), but the 428

molecular mechanisms involved are unclear. In the present work, we present conclusive data 429

demonstrating that low temperature can transcriptionally modulate natural peel degreening in 430

lemon fruit independently of the ethylene signal. 431


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432

Results obtained in this study demonstrate very clearly that moderately low storage 433

temperatures promoted peel degreening (Fig. 1B). Because of the known involvement of 434

ethylene in citrus degreening (Fig. 1A), peel colour changes that occur during low 435

temperature storage have often been attributed to ethylene signalling, that is, trace levels of 436

physiologically active system I ethylene are thought to be bound in tissues (Goldschmidt et 437

al., 1993; Carmona et al., 2012b). Ethylene-induced degreening is completely inhibited by 438

pre-treatment with 1-MCP (Fig. 1A; Jomori et al., 2003; McCollum and Maul, 2007; Li et al., 439

2016). 1-MCP treatment also inhibits the ripening process in fruit that have a strong 440

requirement for ethylene to ripen (Watkins, 2006). In higher plants, ethylene receptors act as 441

negative regulators (Hua and Meyerowitz, 1998), and their binding by ethylene subjects them 442

to degradation via the ubiquitin-proteasome pathway (Kevany et al., 2007). 1-MCP is 443

assumed to irreversibly bind and phosphorylate ethylene receptors (Kamiyoshihara et al., 444

2012), with a higher affinity than ethylene (Jiang et al., 1999), resulting in relatively stable 445

complexes that suppress ethylene signalling even in the presence of ethylene. If endogenous 446

ethylene was physiologically active, then its action should be suppressed by the application 447

of ethylene antagonists such as 1-MCP. However, it is surprising that peel degreening elicited 448

by low temperature was not abolished by repeated 1-MCP treatments (Fig. 1C), which 449

indicated that it most likely occurred in an ethylene-independent manner. 450

451

Further evidence for this conclusion is the identification of distinct gene sets that are 452

regulated by either ethylene or low temperature in the flavedo of lemon fruit (Fig. 2). 453

Ethylene-specific genes such as ClCLH1 and ClERF114 were not differentially expressed 454

during low temperature storage (Fig. 3C, 7B), which implies that ethylene signalling was 455

non-functional in stored fruit. Additionally, ethylene treatment did not show any significant 456

effect on the expression of another distinct gene set that were influenced by low temperature, 457

including ClCHYb1 and ClERF3 (Fig. 4C, 7C). This is perhaps the most direct evidence for 458

an ethylene-independent modulation of peel degreening by low temperature. Although the 459

third gene set, including ClPPH, ClLHCB2, ClPSY1, ClLCYb2a, ClNCED5 and ClbHLH25 460

were differentially regulated by either ethylene or low temperature (Fig. 3C, 4C, 5B, 6B, 7D), 461

their stimulation by low temperature was not altered by repeated 1-MCP treatments, 462

excluding any likelihood of ethylene involvement during storage. 463

464


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The degreening observed in lemon fruit exposed to ethylene is, in all likelihood, due to a 465

reduction in peel chlorophyll content (Fig. 3A), which could be attributed to the upregulation 466

of ClCLH1 and ClPPH (Fig. 3C). Ethylene-induced peel chlorophyll degradation in citrus 467

fruit has also been linked to increased transcript levels of homologues of ClCLH1 (Jacob-468

Wilk et al., 1999; Shemer et al., 2008; Yin et al., 2016), and ClPPH (Yin et al., 2016). 469

During storage, however, the minimal expression levels of ClCLH1 excluding any possibility 470

that it might be involved in low temperature-triggered chlorophyll degradation. Instead, the 471

degradation of chlorophylls caused by low temperature can be attributed to the ethylene-472

independent upregulation of ClPPH, which is known to encode an enzyme with a similar 473

dephytilation activity as CLH (Schelbert et al., 2009). 474

475

The peel carotenoid content decreased upon degreening in response to both ethylene and low 476

temperature (Fig. 4A). This decrease is not uncommon as previous studies have also 477

demonstrated that the peel content of carotenoids, especially lutein, in lemon fruit decreased 478

during maturation (Kato, 2012; Conesa et al., 2019). Nevertheless, the yellowish appearance 479

of degreened lemon fruit (Fig. 1) could be attributed to the small but significant levels of 480

lutein, β-carotene and α-carotene (Fig. 4A), which might be intensified by their unmasking 481

brought about by the loss of chlorophyll. Changes in peel carotenoid content are initiated by 482

the expression of various carotenoid metabolism-related genes which can be stimulated by 483

either ethylene or low temperature (Fig. 4B; Matsumoto et al., 2009; Rodrigo and Zacarias, 484

2007). In this study, however, it appears that carotenoid metabolism-associated genes such as 485

ClPSY1, ClLCYb2a and ClCHYb1 are also transcriptionally modulated by low temperature 486

independently of ethylene. 487

488

The degradation of chlorophylls caused by exposure to either ethylene or low temperature 489

could also be facilitated by changes in photosystem proteins. The disruption of pigment-490

protein complexes is thought to be a crucial step in the chlorophyll degradation pathway 491

(Barry, 2009). Consequently, the stay-green protein (SGR), which encodes a Mg-dechelatase 492

(Shimoda et al., 2016), has been shown to aid the dis-aggregation of photosystem proteins, 493

particularly the light-harvesting chlorophyll a/b binding (CAB) complex (Jiang et al., 2011; 494

Sakuraba et al., 2012). Because photosystem proteins bind pigments, a large drop in their 495

transcripts caused by ethylene or low temperature (Fig. 5) would possibly favour the 496

accumulation of free chlorophylls that can easily be accessed by degradatory enzymes. Peng 497

et al. (2013) also reported that the transcript levels of CitCAB1 and CitCAB2 drastically 498


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16

decreased during ethylene-induced and natural peel degreening in ‘Ponkan’ mandarins. 499

However, the results of the present study suggest that the decrease in the expression of 500

photosystem-encoding genes during natural peel degreening could be stimulated by low 501

temperature independently of ethylene. 502

503

Besides ethylene, various phytohormones such as ABA, GA and JA have been implicated in 504

the peel colour changes that occur during citrus fruit maturation. Peel degreening was shown 505

to be accompanied by an increase in ABA content (Goldschmidt et al., 1973), as well as in 506

the expression of ABA biosynthetic and signalling elements (Rodrigo et al., 2006; Kato et al., 507

2006). In addition, exogenous ABA accelerated fruit ripening and enhanced fruit colour 508

development (Wang et al., 2016), while ABA-deficient citrus mutants showed a delay in the 509

rate of peel degreening (Rodrigo et al., 2003). In this study, ABA levels increased in 510

ethylene-treated and low temperature-stored fruit (Fig. 6C), accompanied by an increase in 511

the expression of ABA biosynthetic and signalling genes (Fig. 6A, B). These findings, 512

together with previous reports, suggest that ABA has a positive regulatory role in either 513

ethylene-induced or low temperature-modulated peel degreening in lemon fruit. GA, on the 514

other hand, is known to retard peel colour change. GA application on green citrus fruit was 515

shown to cause a significant delay in peel colour break (Alós et al., 2006; Rodrigo and 516

Zacarias, 2007; Rios et al., 2010). It is therefore logical that the transcript levels of the GA 517

biosynthetic gene (ClGA20ox2) would decrease, whereas those of GA degradatory genes 518

(ClGA2ox4 and GA2ox8) would increase during peel degreening caused by either ethylene or 519

low temperature (Fig. 6A). JAs have thus far been studied in the context of plant adaptive 520

responses to various biotic and abiotic stresses (Zhang et al., 2019). However, JA has also 521

been shown to promote fruit ripening in citrus (Zhang et al., 2014), strawberry (Concha et al., 522

2013) and tomato (Liu et al., 2012). This is consistent with the present findings as lemon fruit 523

degreening was accompanied by an increase in the levels of JA-Ile (Fig. 6C), which is the 524

active conjugate of JA. Additionally, the expression of a large number of JA biosynthetic and 525

signalling-related genes were independently upregulated by either ethylene treatment or low 526

temperature (Fig. 6A). 527

528

Developmentally regulated plant processes such as peel degreening are typically influenced 529

by TFs. Various TFs such as AtNAC046, AtPIF4, AtPIF5, AtORE1 and AtEIN3 were shown 530

to significantly enhance leaf senescence in Arabidopsis by promoting the activity of 531

chlorophyll degradation-related genes (Song et al., 2014; Qiu et al., 2015; Zhang et al., 2015; 532


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17

Oda-Yamamizo et al., 2016). In broccoli, MYB, bHLH, and bZIP gene families were 533

associated with chlorophyll metabolism while NACs and ERFs regulated carotenoid 534

biosynthesis (Luo et al., 2019). CitERF6 and CitERF13 have also been associated with 535

chlorophyll degradation during ethylene-induced and natural peel degreening in citrus (Yin et 536

al., 2016; Li et al., 2019). In the present work, the expression patterns of genes encoding a 537

wide range of TF families suggested that ethylene-induced and low temperature-modulated 538

peel degreening pathways were distinct in lemon fruit (Fig. 7). Therefore, ethylene-induced 539

degreening is most likely to be regulated by ethylene-specific TFs such as ClERF114 (Fig. 540

7B) and shared TFs such as ClbHLH25 (Fig. 7D). In contrast, low temperature-modulated 541

degreening could be regulated by specific TFs such as ClERF3 (Fig. 7C), as well as shared 542

ones such as ClbHLH25 (Fig. 7D). 543

544

In this study, low temperature appears to also play a prominent role in natural peel 545

degreening during on-tree lemon fruit maturation. Peel degreening and the associated 546

reduction in the content of chlorophylls and carotenoids coincided with gradual drops in 547

minimum environmental temperatures, to below 13ºC (Fig. 8A–C). Similar to our study, 548

previous studies have also demonstrated that peel degreening in most citrus fruit progresses 549

as the environmental temperature decreases (Manera et al., 2012; Manera et al., 2013; 550

Rodrigo et al., 2013; Conesa et al., 2019). It is intriguing that ClCLH1 and ClERF114, which 551

were earlier shown to exhibit an ethylene-specific pattern (Fig. 3C, 7B) did not show any 552

significant changes in expression during on-tree peel degreening (Fig. 8D, F). Earlier studies 553

have also demonstrated that ClCLH1 homologues in other citrus fruit exhibit a dramatic 554

induction in response to ethylene treatment, yet lack a measurable expression increase during 555

natural degreening (Jacob-Wilk et al., 1999; Yin et al., 2016). Here, we suggest that this 556

discrepancy could be due to a lack of a functional ethylene signalling during on-tree 557

maturation, given that ClCLH1 is only upregulated in the presence of ethylene (Fig. 3C). In 558

contrast, genes that responded to low temperature during storage such as ClPPH, ClLHCB2, 559

ClPSY1, ClLCYb2a, ClCHYb1, ClERF3, ClbHLH25 also exhibited similar expression 560

patterns during on-tree maturation (Fig. 7D–F), indicating that they were involved in the on-561

tree peel degreening processes. These similarities between low temperature-induced and on-562

tree gene expression patterns, coupled with their dissimilarities to ethylene-induced changes, 563

provide clear evidence to suggest that on-tree peel degreening responses are modulated by 564

low temperature independently of ethylene in lemon fruit. 565

566


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It is also important to note that many genes were differentially expressed in fruit at 5ºC, yet 567

the peel degreening rate was significantly slower than in fruit at 10ºC, 15ºC and 20ºC. This is 568

probably due to low activity of peel degreening-associated enzymes at 5ºC, as low 569

temperature is known to decrease enzyme activity in plants (Jin et al., 2009; Yun et al., 2012). 570

571

The regulation of fruit ripening by low temperature is not unique to citrus fruit. Previous 572

studies have also demonstrated a role for low temperature, either independently or in concert 573

with ethylene, in the regulation of fruit ripening in multiple fruit species such as kiwifruit 574

(Mworia et al., 2012; Asiche et al., 2017; Asiche et al., 2018; Mitalo et al., 2018a; Mitalo et 575

al., 2019a; Mitalo et al., 2019b), pears (El-Sharkawy et al., 2003; Mitalo et al., 2019c) and 576

apples (Tacken et al., 2010). From an ecological perspective, the primary purpose of fruit 577

ripening is to make fruit attractive to seed-dispersing organisms. To ensure their future 578

survival, most temperate fruit are faced with the challenge of dispersing their seeds in time 579

before the onset of harsh winter conditions. Therefore, environmental temperature drops 580

associated with autumn might provide an alternative stimulus for fruit ripening induction in 581

fruits that lack a functional ethylene signalling pathway during maturation, like citrus. 582

583

In conclusion, the present work provides a comprehensive overview of ethylene- and low 584

temperature-induced peel degreening responses during maturation in lemon fruit by 585

comparing physiochemical changes and corresponding transcriptome changes. Both ethylene 586

and low temperature promote peel degreening by inducing transcriptome changes associated 587

with chlorophyll degradation, carotenoid metabolism, photosystem disassembly, 588

phytohormones and TFs. However, blocking ethylene signalling by repeated 1-MCP 589

treatments does not eliminate low temperature-induced changes. On-tree peel degreening, 590

which typical occurs as minimum environmental temperature drops, corresponds with the 591

differential regulation of low temperature-regulated genes whereas genes that uniquely 592

respond to ethylene do not exhibit any significant expression changes. These data suggest 593

that low temperature plays a prominent role in promoting natural peel degreening both on and 594

off the tree. In our further studies, we aim to identify the direct and indirect targets of low 595

temperature-regulated transcripts that have been uncovered in this study towards elaboration 596

of the molecular bases for low temperature modulation of peel degreening and fruit ripening 597

in general. 598

599

Supplementary data 600


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19

Fig. S1: Chromatograms showing the identified carotenoids. 601

Table S1: Liquid chromatography conditions for phytohormone analysis. 602

Table S2: Parameters for LC-ESI-MS/MS analysis of phytohormones. 603

Table S3: Primer sequences used for RT-qPCR. 604

Table S4: DEGs exclusively responding to ethylene. 605

Table S5: DEGs responding to ethylene and 5ºC. 606

Table S6: DEGs responding to ethylene and 15ºC. 607

Table S7: DEGs responding to ethylene, 5ºC and 15ºC. 608

Table S8: DEGs exclusively influenced by 5ºC. 609

Table S9: DEGs influenced only by 5ºC and 15ºC. 610

Table S10: DEGs exclusively influenced by 15ºC. 611

Table S11: Selected DEGs associated with peel degreening. 612

613

Acknowledgements 614

This study was supported in part by a Grant-in-Aid for Scientific Research (grant no. 615

24380023 and 16H04873) from the Japan Society for the Promotion of Science, and by the 616

Joint Usage/Research Centre, Institute of Plant Science and Resources, Okayama University. 617


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Figure legends

Fig. 1. Promotion of peel degreening in detached lemon fruit by ethylene and low

temperature. (A) Peel colour changes in response to ethylene and 1-methylcyclopropene (1-

MCP) treatments. Control: non-treated; ET: continuously treated with 100 µLL–1 ethylene; 1-

MCP: treated with 2 µLL–1 1-MCP twice a week; 1-MCP+ET: pre-treated with 2 µLL–1 1-

MCP for 12 h before continuous treatment with 100 µLL–1 ethylene. All treatments were

carried out at 25ºC. (B) Peel colour changes during storage at 5ºC, 10ºC, 15ºC, 20ºC and

25ºC in an ethylene-free environment. (C) Effect of 1-MCP treatment on peel colour changes

during storage at 5ºC, 15ºC and 25ºC. Treatments with 1-MCP (2 µLL–1) were carried out

twice a week to block endogenous ethylene action. Data points in represent the mean (±SE)

of five fruit and different letters indicate significant differences in ANOVA (Tukey’s test, p <

0.05).

Fig. 2. Global transcriptome changes induced by ethylene treatment and low

temperature in the flavedo of detached lemon fruit. (A) Number of genes differentially

expressed in response to ethylene treatment and low temperature storage. (B) Clustering

analysis and heatmap of expression measures of DEGs detected in each of the experimental

conditions. (C) and (D) Venn diagrams showing the number of shared and unique genes up-

and down-regulated by ethylene, 5ºC and/or 15ºC. ET – ethylene.

Fig. 3. Changes in chlorophyll content and associated gene expression upon exposure to

ethylene or different storage temperatures. (A) Effect of ethylene and storage temperature

on the content of chlorophyll a and chlorophyll b. (B) Heatmap showing identified DEGs

associated with chlorophyll metabolism in fruit exposed to ethylene and low temperature.

Colour panels indicate the log2 value of fold change for ET (ethylene vs. control), 5ºC vs.

25ºC and 15ºC vs. 25ºC. (C) RT-qPCR analysis of chlorophyllase 1 (ClCLH1) and

pheophytinase (ClPPH) selected from (B) in fruit exposed to ethylene and different storage

temperatures. Data are means (±SE) of three biological replicates (three fruit). Different

letters indicate significant differences in ANOVA (Tukey test, p < 0.05).

Fig. 4. Changes in the content of carotenoids and expression of associated metabolism

genes upon exposure to ethylene and different storage temperatures. (A) Effect of

ethylene and storage temperature on the content of lutein, β-carotene and α-carotene. (B)

Heatmap of identified DEGs associated with carotenoid metabolism in fruit exposed to


https://doi.org/10.1101/855775


29

ethylene and low temperature. Colour panels indicate the log2 value of fold change for ET

(ethylene vs. control), 5ºC vs. 25ºC and 15ºC vs. 25ºC. (C) RT-qPCR analysis of phytoene

synthase 1 (ClPSY1), lycopene cyclase 2a (ClLCYb2a) and β-carotene hydroxylase 1

(ClCHYb1) selected from (B) in fruit exposed to ethylene and different storage temperatures.

Data are means (±SE) of three biological replicates (three fruit). Different letters indicate

significant differences in ANOVA (Tukey test, p < 0.05).

Fig. 5. Changes in the expression of genes encoding photosystem proteins in response to

ethylene and different storage temperatures. (A) Heatmap of identified DEGs encoding

photosystem proteins in fruit exposed to ethylene and low temperature. Colour panels

indicate the log2 value of fold change for ET (ethylene vs. control), 5ºC vs. 25ºC and 15ºC vs.

25ºC. (B) RT-qPCR analysis of light harvesting complex 2 (ClLHCB2) selected from (A) in

fruit exposed to ethylene and different storage temperatures. Data are means (±SE) of three

biological replicates (three fruit). Different letters indicate significant differences in ANOVA

(Tukey test, p < 0.05).

Fig. 6. Levels of phytohormones and the expression of associated genes in the flavedo of

detached lemon fruit. (A) Heatmap showing DEGs encoding proteins associated with

phytohormone biosynthesis and signalling in fruit exposed to ethylene and low temperature.

Colour panels indicate the log2 value of fold change for ET (ethylene vs. control), 5ºC vs.

25ºC and 15ºC vs. 25ºC. (B) RT-qPCR analysis of the ABA biosynthetic gene, 9-cis-

epoxycarotenoid dioxygenase 5 (ClNCED5), selected from (A) in fruit exposed to ethylene

and different storage temperatures. (C) Levels of ABA and JA-Ile in lemon fruit treated with

ethylene and after storage at specified temperatures. Data are means (±SE) of three biological

replicates (three fruit). Different letters indicate significant differences in ANOVA (Tukey

test, p < 0.05).

Fig. 7. Changes in expression of transcription factor-encoding genes. (A) Heatmap

showing identified DEGs encoding various transcription factors in fruit exposed to ethylene

and low temperature. Colour panels indicate the log2 value of fold change for ET (ethylene vs.

control), 5ºC vs. 25ºC and 15ºC vs. 25ºC. (B), (C) and (D) RT-qPCR analysis of ClERF114,

ClERF3 and ClbHLH25 in response to exogenous ethylene and different storage temperatures.

Data are means (±SE) of three biological replicates (three fruit). Different letters indicate

significant differences in ANOVA (Tukey test, p < 0.05).


https://doi.org/10.1101/855775


30

Fig. 8. Peel colour changes and gene expression analysis in lemon fruit during on-tree

maturation. (A) Appearance and citrus colour index of representative fruit at different

developmental stages alongside changes in minimum environmental temperatures. Data for

minimum temperature were accessed from the website of Japan Meteorological Agency

(http://www.data.jma.go.jp/obd/stats/etrn/view/daily_s1.php?prec_no=72&block_no=47891

&year=2014&month=12&day=&view=p1). (B) Chlorophyll a and chlorophyll b contents at

different developmental stages. (C) Levels of lutein, α-carotene and β-carotene at different

developmental stages. RT-qPCR analysis of selected genes associated with chlorophyll

metabolism and photosystem proteins (D), carotenoid metabolism (E), and transcription

factors (F) at different developmental stages. Data points represent the mean (±SE) of five

fruit and different letters indicate significant differences in ANOVA (Tukey’s test, p < 0.05).


https://doi.org/10.1101/855775


Fig. 1. Promotion of peel degreening in detached lemon fruit by ethylene and low temperature. (A) Peel colour changes in response toethylene and 1-methylcyclopropene (1-MCP) treatments. Control: non-treated; ET: continuously treated with 100 µLL–1 ethylene; 1-MCP:treated with 2 µLL–1 1-MCP twice a week; 1-MCP+ET: pre-treated with 2 µLL–1 1-MCP for 12 h before continuous treatment with 100 µLL–1ethylene. All treatments were carried out at 25ºC. (B) Peel colour changes during storage at 5ºC, 10ºC, 15ºC, 20ºC and 25ºC in an ethylene-free environment. (C) Effect of 1-MCP treatment on peel colour changes during storage at 5ºC, 15ºC and 25ºC. Treatments with 1-MCP (2µLL–1) were carried out twice a week to block endogenous ethylene action. Data points in represent the mean (±SE) of five fruit and differentletters indicate significant differences in ANOVA (Tukey’s test, p < 0.05).

A

C

0 2 4 8

Control

ET

1-MCP

1-MCP+ET

Days after harvest

-15

-10

-5

0

5

0 2 4 8

Control1-MCP ET1-MCP+ET

Days after harvest

Citr

us c

olou

r ind

ex (1

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a/L•

b)

a

b

c

de

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e

ee

5ºC

10ºC

20ºC

25ºC

0 7 14 21 28 35 42

15ºC

Days in storage

-15

-10

-5

0

5

0 7 14 21 28 35 42

5ºC

10ºC

15ºC

20ºC

25ºC

Days in storage

Citr

us c

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b)aa

b

c

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d

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15ºC+1-MCP

5ºC+1-MCP

0 7 14 21 28 35 42

25ºC+1-MCP

Days in storage -15

-10

-5

0

5

0 7 14 21 28 35 42

5ºC+1-MCP

15ºC+1-MCP

25ºC+1-MCP

Days in storage

Citr

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)

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bb

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Control ET 15ºC 25ºC5ºC

-2 -1 0 1 2

Z-score210-1-2

Height

1331

238

369

343

607

ET vs. Control

592

27030

22274

124

37

ET vs. Control

700

36525

35950

208

49

A B

C

Fig. 2. Global transcriptome changes induced by ethylene treatment and low temperature in the flavedo of detached lemon fruit. (A)Number of genes differentially expressed in response to ethylene treatment and low temperature storage. (B) Clustering analysis andheatmap of expression measures of DEGs detected in each of the experimental conditions. (C) and (D) Venn diagrams showing the numberof shared and unique genes up- and down-regulated by ethylene, 5ºC and/or 15ºC. ET – ethylene.

0

200

400

600

800

1000

1200

1400

1600

ET vs. Control 5 ºC vs. 25 ºC 15 ºC vs. 25 ºC

Up-regulated

Down-regulated

Num

ber

of D

EGs

D

A

B

C

< -6 > 6

ET 5ºC 15ºCAnnotationSymbolTranscriptChlorophyllase 1Chlorophyllase 2

Geranylgeranyl reductase

Pheophytinase

Magnesium-chelatase subunit chlH

Pheophorbide a oxygenase 1

Pheophorbide a oxygenase 2

Red chlorophyll catabolite reductase

Protochlorophyllide reductase

Chlorophyllase 3

CLH1CLH2CLH3

PPH

GGR

CHLH

PaO1

PaO2

ACD2

PORA

Ciclev10021103m.gCiclev10021095m.gCiclev10005453m.g

Ciclev10031731m.g

Ciclev10008936m.g

Ciclev10004154m.g

Ciclev10014840m.g

Ciclev10014836m.g

Ciclev10026041m.g

Ciclev10008520m.g

Fig. 3. Changes in chlorophyll content and associated gene expression upon exposure to ethylene or different storagetemperatures. (A) Effect of ethylene and storage temperature on the content of chlorophyll a and chlorophyll b. (B)Heatmap showing identified DEGs associated with chlorophyll metabolism in fruit exposed to ethylene and lowtemperature. Colour panels indicate the log2 value of fold change for ET (ethylene vs. control), 5ºC vs. 25ºC and 15ºCvs. 25ºC. (C) RT-qPCR analysis of chlorophyllase 1 (ClCLH1) and pheophytinase (ClPPH) indicated by black arrowsin (B) in fruit exposed to ethylene and different storage temperatures. Data are means (±SE) of three biologicalreplicates (three fruit). Different letters indicate significant differences in ANOVA (Tukey test, p < 0.05).

0

50

100

150

200

250

At-h

arve

st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

ClCLH1

0d 4 d 28 d

Rel

ativ

e ex

pres

sion

d

a

b

cd

c cd c c c c

0

20

40

60

80

100

120

140

At-h

arve

st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

ClPPH

0d 4 d 28 d

ab

d d d

ab ab

ab

b b

cd d

0 d

0

20

40

60

80

100

At-h

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st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

Chl a

0

20

40

60

80

100

At-h

arve

st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

Chl b

Pigm

ent c

onte

nt (µ

g•g–1

)

0 d 4 d 28 d 4 d 28 d

abc

abc

de

bbc

abc

c

d dd

abcab

bc

d

bc

d

c

de e

cc

c

0 d

A

B

C

Fig. 4. Changes in the content of carotenoids and expression of associated metabolism genes upon exposure to ethylene anddifferent storage temperatures. (A) Effect of ethylene and storage temperature on the content of lutein, β-carotene and α-carotene. (B) Heatmap of identified DEGs associated with carotenoid metabolism in fruit exposed to ethylene and lowtemperature. Colour panels indicate the log2 value of fold change for ET (ethylene vs. control), 5ºC vs. 25ºC and 15ºC vs. 25ºC.(C) RT-qPCR analysis of phytoene synthase 1 (ClPSY1), lycopene cyclase 2a (ClLCYb2a) and β-carotene hydroxylase 1(ClCHYb1) selected from (B) in fruit exposed to ethylene and different storage temperatures. Data are means (±SE) of threebiological replicates (three fruit). Different letters indicate significant differences in ANOVA (Tukey test, p < 0.05).

< -6 > 6

ET 5ºC 15ºCAnnotationSymbolTranscriptGeranylgeranyl pyrophosphate synthase 7aGeranylgeranyl pyrophosphate synthase 1

Phytoene desaturase 1

Geranylgeranyl pyrophosphate synthase 2

Lycopene cyclase 2a

Carotenoid cleavage dioxygenase 1

Phytoene synthase 1

Phytoene synthase 2

Phytoene desaturase 2

Geranylgeranyl pyrophosphate synthase 7b

Violaxanthin de-epoxidase

Beta-carotene hydroxylase

Beta hydroxylase

GGPS7aGGPS1

GGPS7b

GGPS2

PDS1

LCYb2a

CCD1

PSY1

PSY2

PDS2

VDE

CHYb1

CHYb2


Ciclev10002259m.g

Ciclev10028457m.g

Ciclev10028245m.g

Ciclev10031014m.g

Ciclev10011841m.g

Ciclev10015582m.g

Ciclev10031587m.g

Ciclev10025312m.gCiclev10022121m.g

Ciclev10005481m.g

0

5

10

15

20

25At

-har

vest

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

Lutein

0

2

4

6

8

At-h

arve

st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

β-Carotene

0

1

2

3

4

At-h

arve

st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

α-Carotene

0 d 4 d 28 d 0 d 4 d 28 d 0 d 4 d 28 d

Pigm

ent c

onte

nt (µ

g•g–1

)

a

ab

bc

b b

ab

b

cc

c

ab

ab ab

abc

c

bc

c

bc

c

c cc

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ab

bb b

ab

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0 d 4 d 28 d

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10

15

20

25

At-h

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Con

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ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

ClCHYb1

dd

d d

b

b

ab

c

c c

d d

0

20

40

60

80

100

At-h

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st

Con

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ET1-

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P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

ClLCYb2a

gh

ab

e

bc

c

b

d

b

c

f f

0 d 4 d 28 d

0

5

10

15

20

25

30

35

40

At-h

arve

st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

ClPSY1

d

c

ab

d

c c

abc

bb

cc

c

0 d 4 d 28 d

< -6 > 6

ET 5ºC 15ºC

Antenna complex

Photosynthetic component

Photosystem I

Photosystem II

PS-I light harvesting complex gene 6PS-I light harvesting complex gene 5

Light harvesting complex PS-II

PS-I light harvesting complex gene 1


Light harvesting protein complex I

Light harvesting protein complex II




Light harvesting complex PS-II 5

PS-II light harvesting complex gene 2

Chlorophyll A-B binding protein

Chlorophyll A-B binding protein 2

Chlorophyll A-B binding protein 2Light-harvesting binding protein 3

LHCA6LHCA5LHCA3

LHCA1

LHCB6

LHCA2

LHCA4

LHCB1

LHCB4

LHCB4

LHCB5

LHCB2

PSBS

CAB2

CAB2LHCB3


Ciclev10032568m.g

Ciclev10029116m.g

Ciclev10031886m.g

Ciclev10021861m.g

Ciclev10016287m.g

Ciclev10032377m.g

Ciclev10012370m.g


Ciclev10002099m.g

Ciclev10016074m.g


AnnotationSymbolTranscript

Photosystem I subunit F

Photosystem I P subunit

Photosystem I subunit K

Photosystem I subunit D-2

Photosystem I subunit E-2

PS-I reaction center subunit PSI-N

Photosystem I subunit G

Photosystem I subunit D-2

Photosystem I subunit l

Photosystem I subunit H2

Photosystem I subunit O

PSAF

PSAP

PSAK

PSAD-2

PSAE-2

PSAN

PSAG

PSAD-2

PSAL

PSAH-2

PSAO

Ciclev10016540m.g

Ciclev10022571m.g

Ciclev10017170m.g

Ciclev10005892m.g

Ciclev10022750m.g

Ciclev10002687m.g

Ciclev10009769m.g

Ciclev10022248m.g

Ciclev10016637m.g

Ciclev10026722m.g

Ciclev10029515m.g

Photosystem II subunit R

Photosystem II reaction center WPhotosystem II 5 kD protein

Photosystem II reaction center PsbPPhotosystem II subunit Q-2

PS-II stability/assembly factorHigh chlorophyll fluorescent 107Photosystem II reaction center W

Photosystem II reaction center PSB28Photosystem II subunit T

Photosystem II family

PSBR

PSBWPSBLPSBP

PSBQ-2

HCF136HCF107PSBWPSB28PSBTNPSB7

Ciclev10022793m.g

Ciclev10024455m.gCiclev10033132m.gCiclev10002020m.gCiclev10016525m.g

Ciclev10031672m.gCiclev10004521m.gCiclev10029551m.gCiclev10022322m.gCiclev10017247m.gCiclev10016927m.g

Photosystem II subunit X

Photosystem II BYPhotosystem II subunit X

Photosystem II subunit O-2Photosystem II subunit P-1

Mog1/PsbP/DUF1795-like PS-II reaction centerMog1/PsbP/DUF1795-like PS-II reaction centerMog1/PsbP/DUF1795-like PS-II reaction center

PSBX

PSBYPSBX

PSBO-2PSBP-1

PPD5PPD5PPD5

Ciclev10009914m.g



AB

Fig. 5. Changes in the expression of genesencoding photosystem proteins in response toethylene and different storage temperatures. (A)Heatmap of identified DEGs encodingphotosystem proteins in fruit exposed to ethyleneand low temperature. Colour panels indicate thelog2 value of fold change for ET (ethylene vs.control), 5ºC vs. 25ºC and 15ºC vs. 25ºC. (B) RT-qPCR analysis of light harvesting complex 2(ClLHCB2) indicated by a black arrow in (A) infruit exposed to ethylene and different storagetemperatures. Data are means (±SE) of threebiological replicates (three fruit). Different lettersindicate significant differences in ANOVA (Tukeytest, p < 0.05).

0

1

2

3

4

At-h

arve

st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

ClLHCB2

0 d 4 d 28 d

Rel

ativ

e ex

pres

sion

a

abab

cc

d

dd d

dd

d

A B

C

ET 5ºC 15ºCAnnotationSymbolTranscriptHormone1-amino-cyclopropane-1-carboxylate synthase 81-amino-cyclopropane-1-carboxylate synthase 1

1-amino-cyclopropane-1-carboxylate oxidase 4

Ethylene response sensor 1

Ethylene receptor 2

1-amino-cyclopropane-1-carboxylate synthase 10

ACS8ACS1

ACS10

ACO4

ERS1

ETR2


Ciclev10015962m.g

Ciclev10019132m.g

Ciclev10004385m.g

Ethylene

Allene oxide cyclase 4

Lipoxygenase 2Lipoxygenase 1

Allene oxide synthase 1Allene oxide synthase 3

Allene oxide cyclase 2Allene oxide cyclase 3

Coronatine insensitive protein 1F-box/RNI-like superfamily proteinJasmonate-zim-domain protein 8

Jasmonate-zim-domain protein 10

AOC4

LOX2LOX1AOS1AOS3

AOC2AOC3COI1COI1JAZ8

JAZ10

Jasmonate-zim-domain protein 1Jasmonate-zim-domain protein 1Jasmonate-zim-domain protein 1Jasmonate-zim-domain protein 3

JAZ1JAZ1JAZ1JAZ3

Ciclev10012528m.g




Jasmonate

Nine-cis-epoxycarotenoid dioxygenase 5Nine-cis-epoxycarotenoid dioxygenase 4Nine-cis-epoxycarotenoid dioxygenase 4

Abscisic acid repressor 1

NCED5NCED4NCED4ABR1


ABA

Indole-3-acetic acid inducible 29

Flavin-containing monooxygenaseFlavin-containing monooxygenaseIndole-3-acetic acid inducible 19Indole-3-acetic acid inducible 14

Indoleacetic acid-induced protein 16Indoleacetic acid-induced protein 8Indoleacetic acid-induced protein 8

Indole-3-acetic acid inducible 14Auxin-induced protein 13

AUX/IAA transcriptional regulator

IAA29

YUC10YUC10IAA19IAA14

IAA16IAA8IAA8

IAA14IAA13

AUX/IAA2

Auxin-responsive GH3Auxin-responsive GH3

Auxin-responsive factor AUX/IAA-relatedAuxin response factor 18

GH3GH3

ARF3ARF18

Ciclev10009245m.g




SAUR-like auxin-responsive protein

Auxin response factor 4Auxin response factor 8

SAUR-like auxin-responsive proteinSAUR-like auxin-responsive protein

SAUR-like auxin-responsive proteinSAUR-like auxin-responsive proteinSAUR-like auxin-responsive proteinSAUR-like auxin-responsive proteinSAUR-like auxin-responsive protein

Auxin efflux carrier

SAUR50

ARF4ARF8

SAUR32SAUR71

SAUR24SAUR20SAUR76SAUR32SAUR21

PIL7

Auxin efflux carrierAuxin efflux carrier

PIL1PIL3

Ciclev10029633m.g




Auxin

< -6 > 6

Gibberellin-20-oxidase 2GA20ox2Gibberellin-2-oxidase 4Gibberellin-2-oxidase 8

GA2ox4GA2ox8


Gibberellin

Fig. 6. Levels of phytohormones and the expression of associated genes in the flavedo of detached lemon fruit. (A) Heatmap showing DEGsencoding proteins associated with phytohormone biosynthesis and signalling in fruit exposed to ethylene and low temperature. Colour panelsindicate the log2 value of fold change for ET (ethylene vs. control), 5ºC vs. 25ºC and 15ºC vs. 25ºC. (B) RT-qPCR analysis of the ABA biosyntheticgene, 9-cis-epoxycarotenoid dioxygenase 5 (ClNCED5), indicated by a black arrow in (A) in fruit exposed to ethylene and different storagetemperatures. (C) Levels of ABA and JA-Ile in lemon fruit treated with ethylene and after storage at specified temperatures. Data are means (±SE)of three biological replicates (three fruit). Different letters indicate significant differences in ANOVA (Tukey test, p < 0.05).

0

5

10

15

20

25

30

35

At-harvest

Con

trol

ET 5ºC

10ºC

15ºC

20ºC

25ºC

0 d 4 d 28 d

0

500

1000

1500

2000

2500

3000

3500

At-harvest

Con

trol

ET 5ºC

10ºC

15ºC

20ºC

25ºC

0 d 4 d 28 d

JA-I

leco

nten

t (ng

•g–1

)AB

A co

nten

t (ng

•g–1

)

bc

a

bc

dd

ee

aa

de

c

ee

d

ff

0

50

100

150

200

250

At-h

arve

st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

ClNCED5

0 d 4 d 28 d

Rel

ativ

e ex

pres

sion

e

a

ababab

c

dee

de e

d

A B

C

D

Fig. 7. Changes in expression of transcription factor-encoding genes. (A)Heatmap showing identified DEGs encoding various transcription factors infruit exposed to ethylene and low temperature. Colour panels indicate thelog2 value of fold change for ET (ethylene vs. control), 5ºC vs. 25ºC and 15ºCvs. 25ºC. (B), (C) and (D) RT-qPCR analysis of ClERF114, ClERF3 andClbHLH25 in response to exogenous ethylene and different storagetemperatures. Data are means (±SE) of three biological replicates (threefruit). Different letters indicate significant differences in ANOVA (Tukey test,p < 0.05).

ET 5ºC 15ºCAnnotationSymbolTranscriptDomain

Ethylene response factor 023





Ethylene response factor SHINE 2

Apetala 2





ERF023

ERF073

ERF114

ERF017

ERF053

SHN2

AP2

ERF039

ERF1

ERF061

ERF107


Dehydration-responsive element-binding protein 3

Dehydration-responsive element-binding protein 2A

Dehydration-responsive element-binding protein 2D

ERF017

DREB3

DREB2A

DREB2D

Ethylene-responsive element binding factor 13

AP2/B3 transcription factor family protein

AP2/B3 transcription factor family protein

ERF family protein 038

Ethylene-responsive element binding factor 2




C-repeat/DRE binding factor 1

C-repeat/DRE binding factor 2

ERF013

RAVL3

RAVL

ERF038

ERF2

ERF014

ERF110

ERF105

CBF1

CBF2

Ciclev10022410m.g


Ciclev10015144m.g



Ciclev10022025m.g


Ciclev10002294m.gCiclev10026032m.gCiclev10005750m.gCiclev10021923m.gCiclev10022680m.g

AP2/ERF

Basic helix-loop-helix protein 137



bHLH137

bHLH13

bHLH49











bHLH24/SPT

bHLH62

bHLH92

bHLH48

bHLH16/UNE10

bHLH162

bHLH79

bHLH25

bHLH116/ICE1

bHLH74


Ciclev10025735m.g


Ciclev10016466m.gCiclev10021606m.gCiclev10027504m.gCiclev10033497m.gCiclev10001249m.g

bHLH

MYB domain protein 308








MYB-like 41


Transcription factor MYBS3


MYB-like HTH regulator AS1




Protein PHR1-LIKE 3



Protein PHR1-LIKE 1

MYB family transcription factor PHL8

Transcription factor MYBS1

MYB4

MYB308

MYB63

MYB13

MYB306

MYB62

MYB14

MYB77

MYB41

MYB15

MYBS3

MYB111

MYB91/AS1

MYB6

MYB59

PHL8

MYB62

MYB4

MYB16

PHL1

PHL3

MYBS1

Ciclev10023519m.g




Ciclev10031937m.g



MYB

NAC domain containing protein 21/22

NAC domain containing protein 83




NAC domain containing protein 21/22



NAC-like, activated by AP3/PI


NAC21/22

NAC83

NAC25

NAC29

NAC100

NAC21/22

NAC42JUB1

NAC72

NAC47

NAC22


Ciclev10032304m.g



NAC

GRAS family transcription factor 17

GRAS family transcription factor


DELLA protein


DELLA protein GAI


GRAS17

GRAS

GRAS25

GAIP-B

GRAS13

GAI

GRAS19

Ciclev10027940m.gCiclev10007843m.gCiclev10025246m.gCiclev10017466m.gCiclev10011141m.gCiclev10010825m.gCiclev10025169m.g

GRAS

Zinc finger protein GIS3

ZIM17-type zinc finger protein

Zinc finger CCCH domain-containing protein 3

Dof zinc finger protein DOF1.2

C2H2-like zinc finger protein late flowering

Zinc finger protein ZAT11

Zinc finger (C2H2 type) family protein

LIM domain contain protein WLIM1

GATA transcription factor 16



GIS3

ZIM17

C3H3

DOF1.2

LATE

ZAT11

Znf_C2H2

LIM1

GATA16

LIM1

GATA5





GATA22

LIM1

GATA22

GATA21

Ciclev10016426m.g





Zinc finger protein 4

E3 ubiquitin-protein ligase EDA40


Zinc finger CCCH domain-containing protein 18-like

Mini zinc finger 2

B-box type zinc finger protein 30

Zinc finger protein CONSTANS-LIKE 16

B-box zinc finger protein 20

Zinc finger protein CONSTANS-LIKE 16

Zinc finger protein ZAT9

C3H20

ZFP4

EDA40

C3H18

C3H18-like

MIF2

MIP1A

COL16

BBX20

COL16

ZAT9



ZFP4

ZFP2

Ciclev10008633m.g




AN1 domain-containing stress-associated protein 12SAP12Ciclev10012852m.g

Zinc finger

Basic region/leucine zipper motif 53

Homeobox-leucine zipper protein


bZIP53

HB-4

HB-12-like




HB-7

HB-52


Homeobox

< -10 > 10

WRKY transcription factor 31









Squamosa promoter binding protein-like 9

Squamosa promoter binding protein-like 6

WRKY31

WRKY28

WRKY74

WRKY70

WRKY75

WRKY75

WRKY71

WRKY76

WRKY65

SPL9

SPL6

MADS-box protein EJ2

AGAMOUS-like 82

TCP family transcription factor 20


EJ2

AGL82

TCP20

TCP22

Ciclev10014642m.g






TCP8

TCP14


WRKY

MADS-box

TCP

Ethylene response factor 3ERF3Ciclev10010348m.g

0

5

10

15

20

25

30

35

At-h

arve

st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

ClERF114

0 d 4 d 28 d

Rel

ativ

e ex

pres

sion

a

b b bb b b b b b

b b

0

2

4

6

8

10

12

At-h

arve

st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

ClERF3

0 d 4 d 28 d

Rel

ativ

e ex

pres

sion

a a

bc

abc

ab

dd

c

d dd

d

0

100

200

300

400

500

600

At-h

arve

st

Con

trol

ET1-

MC

P+ET 5ºC

5ºC

+1-M

CP

10ºC

15ºC

15ºC

+1-M

CP

20ºC

25ºC

25ºC

+1-M

CP

ClbHLH25

0 d 4 d 28 d

Rel

ativ

e ex

pres

sion a

abab

ab

bcd

bc

ddd

d

e e

Fig. 8. Peel colour changes and gene expression analysis in lemon fruit during on-tree maturation. (A) Appearance and citruscolour index of representative fruit at different developmental stages alongside changes in minimum environmentaltemperatures. Data for minimum temperature were accessed from the website of Japan Meteorological Agency(http://www.data.jma.go.jp/obd/stats/etrn/view/daily_s1.php?prec_no=72&block_no=47891&year=2014&month=12&day=&view=p1). (B) Chlorophyll a and chlorophyll b contents at different developmental stages. (C) Levels of lutein, α-carotene and β-carotene at different developmental stages. RT-qPCR analysis of selected genes associated with chlorophyll metabolism andphotosystem proteins (D), carotenoid metabolism (E), and transcription factors (F) at different developmental stages. Data pointsrepresent the mean (±SE) of five fruit and different letters indicate significant differences in ANOVA (Tukey’s test, p < 0.05).

0

20

40

60

80

100

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

Chl aChl b

171 196 211 230 246 261DAFB

a

d

c

bc

bc

dd e

f

ef

d

c

Pigm

ent c

onte

nt (µ

g•g–

1 )

276

A

C

D

0

5

10

15

20

25

-18

-15

-12

-9

-6

-3

0

3

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

Colour index Temperature

3 Sep. 12 Oct.27 Sep. 30 Oct. 14 Nov. 13 Dec.29 Nov.

171 196 211 230 246 261 276DAFB

Tem

pera

ture

(ºC)

Citr

us c

olou

r ind

ex (1

000•a

/L•b

)

171 196 211 230 246 261 276DAFB

aa

b

b

cc

c

0

5

10

15

20

25

-18

-15

-12

-9

-6

-3

0

33-

Sep

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

Colour index Temperature

3 Sep. 12 Oct.27 Sep. 30 Oct. 14 Nov. 13 Dec.29 Nov.

171 196 211 230 246 261 276DAFB

Tem

pera

ture

(ºC)

Citr

us c

olou

r ind

ex (1

000•a

/L•b

)

171 196 211 230 246 261 276DAFB

aa

b

b

cc

c

B

0

5

10

15

20

25

30

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

Luteinα-Caroteneβ-carotene

Pigm

ent c

onte

nt (µ

g g–

1 )

DAFB 171 196 211 230 246 261 276

a

bb

cd

d

f f

dd

d

de

ee

fe

0

1

2

3

4

5

6

7

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

ClCLH1

0

50

100

150

200

250

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

ClPPH

171 196 211 230 246 261 276 171 196 211 230 246 261 276

Rel

ativ

e ex

pres

sion

b

ababab

abcabc

bc

ab

b

ccd

dee

DAFB

0.0

0.4

0.8

1.2

1.6

2.0

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

ClLHCB2

171 196 211 230 246 261 276

a

bbc

eedd

0

1

2

3

4

5

6

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

ClPSY1

0

5

10

15

20

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

ClLCYb2a

0

2

4

6

8

10

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

ClCHYb1

Rel

ativ

e ex

pres

sion

171 196 211 230 246 261 276DAFB

c

c

c

b

a

cbc

e

e

e

d

c

ab

ab

b

b

a

a

bb b

171 196 211 230 246 261 276171 196 211 230 246 261 276

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

ClERF114

bb

ab

ab

ab

abab

176 196 211 230 246 261 276

0

10

20

30

40

50

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

ClERF3a

c c

b

c

ab

c

171 196 211 230 246 261 276

0

40

80

120

160

200

3-Se

p

27-S

ep

12-O

ct

30-O

ct

14-N

ov

29-N

ov

13-D

ec

ClbHLH25 a

cdd

c

ab

d

171 196 211 230 246 261 276

Rel

ativ

e ex

pres

sion

E

F

Documents

Running title: Low temperature promotes peel degreening in citrus fruit … · Running title: Low temperature promotes peel degreening in citrus fruit Low temperature transcriptionally