Melatonin Antagonizes Jasmonate-Triggered Anthocyanin

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Biotechnology and Biological Transformations

Melatonin antagonizes jasmonate-triggered anthocyanin biosynthesis in Arabidopsis thaliana Yu Ai, and Ziqiang Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01795 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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Journal of Agricultural and Food Chemistry

1

Title: Melatonin antagonizes jasmonate-triggered anthocyanin biosynthesis in

2

Arabidopsis thaliana Yu Ai1 and Ziqiang Zhu1,*

3 1

4

Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life

5

Sciences, Nanjing Normal University, Nanjing 210023, China

6

Running title: Melatonin suppresses anthocyanin induction *

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Corresponding author: Dr. Ziqiang Zhu, email: [email protected]

Keywords: melatonin, jasmonate, anthocyanin, metabolism, Arabidopsis

ACS Paragon Plus Environment

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ABSTRACT: As a plant-specific flavonoid type metabolite, anthocyanin is an

10

important plant-sourced nutrition. Although anthocyanin biosynthesis pathway has

11

been revealed, how to modulate anthocyanin production by endogenous molecules is

12

still elusive. Here, we investigated the role of melatonin in anthocyanin biosynthesis

13

in the reference plant Arabidopsis thaliana and found that melatonin suppresses

14

anthocyanin synthesis. Moreover, melatonin was able to significantly inhibit

15

jasmonate-stimulated anthocyanin production. Unexpectedly, melatonin could not

16

repress the jasmonate-triggered JAZ protein degradation that is a key event for

17

relaying jasmonate signaling. The expression of jasmonate-induced marker genes or

18

other jasmonate-related phenotypes were not discernibly changed in the presence of

19

melatonin. These results indicate that the antagonization of jasmonate-induced

20

anthocyanin synthesis by melatonin does not occur through the abrogation of

21

jasmonate signaling. Furthermore, we found that melatonin does not trigger

22

anthocyanin catabolism. Finally, we supplied anthocyanin biosynthesis precursors to

23

examine their roles in anthocyanin biosynthesis and found that melatonin most likely

24

acts before the dihydrokaempferol production step. Our work illustrates that

25

melatonin plays a negative role in the induction of anthocyanin biosynthesis and sheds

26

new light on the role of melatonin in plant cell metabolism.


2

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27

INTRODUCTION

28

Anthocyanin is a crucial flavonoid type metabolite for controlling plant

29

fertility and protecting plants from environmental stresses [1, 2]. As a solely

30

plant-sourced nutrition, anthocyanin is good for human health [3-6]. Anthocyanin

31

biosynthesis begins with the conversion of L-phenylalanine (L-Phe) to cinnamate,

32

catalyzed by phenylalanine ammonia lyase (PAL). Through a sequential of enzymatic

33

reactions, cinnamate is transformed into coumarate by cinnamate 4-hydroxylase (C4H)

34

and coumarate is further transformed into 4-coumaroyl-CoA by 4-coumaroyl:

35

CoA-ligase (4CL). 4-coumaroyl-CoA and malonyl-CoA are synthesized into

36

naringenin chalcone by chalcone synthase (CHS). Naringenin chalcone is further

37

converted into naringenin by chalcone isomerase (CHI), and naringenin is then

38

transformed

39

Dihydrokaempferol is then transformed into dihydroquercetin by flavonoid

40

3’-hydroxylase (F3’H). Dihydroflavonol reductase (DFR) catalyzes dihydroflavonol

41

into leucoanthocyanidin and then leucoanthocyanidin is converted into anthocyanidins

42

by leucoanthocyanidin dioxygenase (LDOX). Anthocyanidins can be glycosylated by

43

UDP-Glc:flavonoid 3-O-glucosyltransferase (UF3GT) to produce glycosylated

44

anthocyanidins that ultimately form anthocyanin [7-9].

into

dihydrokaempferol

by

flavonone-3-hydroxylase

(F3H).

45

Many environmental or endogenous cues modulate anthocyanin biosynthesis.

46

For example, drought or light treatment induces anthocyanin synthesis to enhance

47

plant fitness [10, 11]. The plant hormone jasmonate also has a positive impact on

48

anthocyanin production [12, 13]. Jasmonate was originally defined as a defense

49

hormone that is necessary for plant resistance to necrotrophic fungi or insect attack

50

[14, 15]. Jasmonate also regulates root elongation [16], root hair development [17],

51

flowering time [18], fruit development [19, 20], fertility and senescence [21-23]. 3



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F-box protein CORONATINE INSENSITIVE 1 (COI1) is the jasmonate receptor that

53

physically interacts with JASMONATE-ZIM-DOMAIN (JAZ) proteins only in the

54

presence of jasmonate [21, 24, 25]. JAZs are then ubiquitinated and degraded through

55

the 26S proteasome [25]. The removal of JAZ repressors releases their repression of

56

JAZ-interacting transcription factors to elicit various jasmonate responses [26]. For

57

example, JAZs interact with WD-Repeat/bHLH/MYB transcription factor complexes

58

to regulate anthocyanin biosynthesis [27]. Among these, MYB75 directly up-regulates

59

the transcription of the ‘late’ anthocyanin biosynthetic genes (DFR, LDOX, and

60

UF3GT) to induce anthocyanin synthesis [28]. A dominant mutation of MYB75 called

61

pap1-D is a T-DNA insertion line that accumulates significantly higher levels of

62

anthocyanin than the wild-type [29].

63

Melatonin (5-methoxy-N-acetyltryptamine) is an indoleamine that was initially

64

discovered in the bovine pineal gland [30]. As a necessary functional molecule,

65

endogenous melatonin exists in bacteria, fungi, animals, and even in plants [31]. It has

66

been reported that in mammalian cells, melatonin regulates circadian rhythm, sleep

67

[32], immunity [33], oncogenesis [34], and scavenging free radicals [35]. Melatonin

68

was identified in plants early in 1995 [36] and has been found to have a range of

69

physiological roles, including regulating cold tolerance [37], drought and salt

70

tolerance [38, 39], seed germination [40], root growth [41], flowering time control

71

[42], fruit ripening [43], and leaf senescence [39].

72

However, the role of melatonin in modulating plant metabolism remains

73

unclear. In this study, we found that melatonin antagonizes jasmonate-induced

74

anthocyanin accumulation in Arabidopsis thaliana by regulating anthocyanin

75

metabolism rather than by affecting jasmonate signaling.

76 4


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MATERIALS AND METHODS

78

Plant Materials and Growth Conditions. Plant materials used in this study were as

79

previously described: coi1-2 [44], pap1-D [29], jazQ [45], aos (Salk_017756) [46],

80

snat (Salk_032239) [47], 35S:JAZ1-GUS [25], MsSNAT-1, and MsSNAT-2 [48]. Seeds

81

were first treated with 5% sodium hypochlorite and 0.1% Triton X-100 solution for 5

82

min, washed five times with sterile water, and then placed on Murashige and Skoog

83

(MS) medium containing certain chemical treatments. Plates were stratified at 4°C for

84

3 d and then placed into a growth chamber (22°C, 80–90 µmolm−2s−1 continuous

85

white light). For photoperiod growth, plates were placed at 22°C under long-day

86

condition (16 h light/8 h dark). For etiolation growth, seeds were placed on MS

87

medium plates including various treatments and stratified at 4°C for three days and

88

then exposed to white light (80–90 µmolm−2s−1) for 3 h to stimulate seed germination.

89

Plates were wrapped with aluminum foil to keep them in complete darkness at 22°C

90

for 7 d.

91 92

Measurement of Anthocyanin Content. Anthocyanin extraction was performed as

93

previously described with minor modifications [49]. Firstly, 10-day-old seedlings

94

were collected and the fresh weight (FW) of each sample was measured. Seedlings

95

were then transferred into one 2 ml centrifuge tube including 1.5 ml of anthocyanin

96

extraction buffer (18% 1-propanol, 1% hydrochloric acid). Centrifuge tubes were

97

boiled for 5 min and then incubated in darkness at 22°C for 12 h. Absorbencies at 535

98

nm (A535) or 650 nm (A650) were determined with a spectrophotometer (Thermo).

99

Anthocyanin contents were calculated by subtracting the A535 value from the A650

100

value and then dividing the result by the FW. Mean values were obtained from three

101

independent replicates. 5



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102 103

Root and Hypocotyl Length Measurement. Seven-day-old Arabidopsis seedlings

104

were imaged and then analyzed with Image J software (http://rsbweb.nih.gov/ij/).

105 106

Quantification of GUS Activity. Seedlings were ground in 200 µl of GUS extraction

107

buffer (50 mM sodium dihydrogen phosphate, 10 mM ethylene diamine tetraacetic

108

acid, 0.1% sodium N-dodecanoylsalcosinate, and 10 mM β-mercaptoethanol) and then

109

centrifuged at 12,000 rpm for 10 min. Next, 2 µl of supernatant was added into 48 µl

110

of

111

4-methyl-umbelliferyl-beta-D-glucuronide and incubated at 37°C for 1 h before the

112

reaction was stopped by adding 1.45 ml of sodium carbonate (0.2 M). Fluorescence

113

intensity was detected with a microplate reader (BioTek) under excitation wavelength

114

(365 nm) and emission wavelength (455 nm).

GUS

extraction

buffer

containing

additional

2

mM

of

115 116

Statistical Method. For all experiments, data were generated from at least three

117

independent biological replicates. Results were analyzed in the STATISTIC 8.0

118

program and evaluated by one-way ANOVA with post-hoc Tukey HSD. Bars marked

119

with different letters differ significantly (Tukey post hoc test, P ≤ 0.05).

120 121

GUS Histochemical Staining. Seedlings were soaked in GUS staining buffer (2 mM

122

potassium

123

5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc), 0.1% Triton X-100, 10

124

mM ethylene diamine tetraacetic acid, and 50 mM sodium phosphate buffer, pH 7.2)

125

at 37°C in darkness for 6 h and then washed with an ethanol series (50%, 70%, 100%)

ferrocyanide,

2

mM

potassium

ferricyanide,

2

mM

6


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126

before finally being recovered with water. Representative GUS staining images were

127

photographed.

128 129

Protein Extraction and Immuno-blotting. Total protein was extracted using a

130

non-grinding method [50]. Briefly, 7-day-old seedlings (10 seedlings in total) were

131

immersed in 150 µl extraction buffer (0.1 M EDTA, pH 8.0, 0.12 M Tris-HCl, pH 6.8,

132

4% SDS, 10% β-mercaptoethanol, 5% glycerol, and 0.005% bromophenol blue) and

133

immediately boiled for 10 min before being centrifuged at 13000 rpm for 5 min.

134

Supernatants were separated on SDS-PAGE gels for immuno-blot analysis. Anti-GUS

135

antibody (Life Technologies) was used to detect JAZ1-GUS protein.

136 137

RNA Isolation and Quantitative Real-time PCR (qRT-PCR). Total RNA was

138

isolated with TRIzol reagent (Invitrogen) and treated with DNase RQ1 (Promega) to

139

remove residual genomic DNA. First-strand cDNA was synthesized from one

140

microgram of the isolated total RNA using a reverse transcription system (Vazyme)

141

following the manufacturer’s instructions. qRT-PCR reactions were performed with

142

AceQ qPCR SYBR Green Master mix (Vazyme) in a LightCycler 96 PCR machine

143

(Roche). Gene expression levels were normalized with the housekeeping gene

144

ACTIN2 (At3g18780).

145 146

RESULTS

147

Melatonin Antagonizes Jasmonate-stimulated Anthocyanin Accumulation.

148

To test whether melatonin is involved in anthocyanin biosynthesis, we initially

149

grew wild-type (Col-0) seedlings under continuous light irradiance in the presence of

150

various concentrations of melatonin and compared their anthocyanin content. We 7



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151

included a variety of melatonin concentrations (ranging from 10 to 500 µM), and

152

found that the extracted anthocyanin content could be slightly reduced when the

153

melatonin concentration reached 100 µM (Fig. 1A, B). However, further increasing

154

melatonin concentrations would gradually rescue the anthocyanin levels (Fig. 1A, B).

155

To exclude the role of light irradiance, we further analyzed anthocyanin levels

156

under photoperiodic conditions. We grew wild-type seedlings under long-day

157

conditions (16 h light/8 h dark), and also included the same melatonin concentrations

158

to compare their anthocyanin content. Similar to the situation under continuous light,

159

melatonin could slightly reduce anthocyanin content except for an extreme high

160

concentration (500 µM) (Fig. 1C, D). Taken together, these results suggest that

161

melatonin has a negative role in anthocyanin biosynthesis, no matter under continuous

162

light or photoperiodic growth condition.

163

Because anthocyanin levels are relatively low under normal growth condition

164

as we illustrated in Figure 1, then we tried to further test melatonin response when

165

anthocyanin levels were initially induced. We choose jasmonate as an inducer to boost

166

anthocyanin biosynthesis and examine the effect of melatonin in this process.

167

Jasmonate-induced anthocyanin accumulation more than 20-fold in the wild-type

168

plants grown under continuous light, but this effect was largely abolished in coi1-2

169

mutants (Fig. 2A). These results are consistent with previous reports that jasmonate

170

stimulates anthocyanin accumulation in a COI1-dependent manner. However, the

171

jasmonate-induced anthocyanin accumulation was attenuated in Col-0 in the presence

172

of melatonin (Fig. 2A, B), suggesting that melatonin has a negative role in

173

jasmonate-triggered anthocyanin accumulation. We also tested this assay under

174

long-day growth condition and further confirmed that melatonin suppressed

175

jasmonate-induced anthocyanin accumulation (Fig. S1). 8


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pap1-D mutants have more anthocyanin under normal growth conditions than

177

wild-type plants, with jasmonate treatment further inducing anthocyanin accumulation

178

in these pap1-D mutants. As in Col-0, melatonin reduced the induction effect of

179

jasmonate in pap1-D lines (Fig. 2A, B).

180

Next, we analyzed the role of melatonin in jazQ mutants that are defective in

181

the expression of five JAZ genes (JAZ1/JAZ3/JAZ4/JAZ9/JAZ10) [45]. Consistent

182

with their roles as jasmonate signaling repressors, jasmonate treatment induced

183

anthocyanin accumulation by more than two-fold in jazQ mutants relative to

184

wild-type. Melatonin treatment, however, further suppressed anthocyanin production

185

in jasmonate-treated jazQ mutants (Fig. 3A, B).

186

To test whether jasmonate biosynthesis is involved in melatonin-mediated

187

suppression of anthocyanin biosynthesis, we selected one jasmonate synthesis mutant

188

aos for further analysis. Allene oxide synthase (AOS) catalyzes the reaction from

189

13-hydroperoxylinoleic acid to 12, 13-epoxyoctadecatrienoic acid that is the key step

190

for

191

13S)-12-oxo-phytodienoic acid (OPDA). Jasmonate treatment induced anthocyanin

192

production in aos mutants whereas melatonin repressed this induction as well (Fig.

193

S2).

the

production

of

the

jasmonate

biosynthesis

precursor

(9S,

194

To exclude the possibility that exogenous melatonin treatment has side effects,

195

we measured the anthocyanin content of melatonin over-producing or defective

196

mutants. Overexpression of the SNAT gene in Arabidopsis (two independent

197

transgenic lines: MsSNAT-1 and MsSNAT-2) causes the over-production of melatonin

198

[48] while snat mutants have less melatonin [47]. Consistent with our previous

199

findings, the anthocyanin levels in either SNAT overexpression lines or mutants were

200

equal to wild-type plants (Fig. 4A, B). However, jasmonate-induced anthocyanin 9



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201

production was attenuated in SNAT overexpression lines (Fig. 4A, B), suggesting that

202

over-production of endogenous melatonin is sufficient for inhibiting the

203

jasmonate-induced anthocyanin biosynthesis.

204 205

Taken

together,

we

concluded

that

melatonin

antagonized

jasmonate-stimulated anthocyanin accumulation.

206 207 208

Melatonin Does Not Abrogate Jasmonate Signaling. As

jasmonate

stimulates

anthocyanin

accumulation

through

the

209

COI1-dependent jasmonate signaling pathway, we speculated that melatonin represses

210

canonical jasmonate signaling. We first checked JAZ1 protein stability through either

211

detection of β-glucuronidase (GUS) activity or immune blotting in JAZ1-GUS

212

transgenic plants. Although JAZ1-GUS fusion protein was gradually decayed under

213

aqueous solution (Fig. 5B-C), most likely due to the flooding stress, jasmonate

214

accelerated its protein turnover. However, the presence of melatonin could not

215

suppress JAZ1 degradation (Fig. 5A–C). Next, we selected six jasmonate-induced

216

marker genes (JAZ1, JAZ2, JAZ3, JAZ5, JAZ9, and JAZ10) to monitor whether

217

melatonin changed their expression levels. These genes were induced by jasmonate,

218

but melatonin did not suppress the induction pattern (Fig. 6). Finally, we tested other

219

characteristic COI1-dependent jasmonate-responsive phenotypes. Jasmonate is known

220

to stimulate anthocyanin biosynthesis and inhibit root and hypocotyl elongation in

221

Arabidopsis seedlings. Although increasing the melatonin concentrations could

222

significantly reduce jasmonate-induced anthocyanin accumulation (Fig. 7A),

223

simultaneous treatment with melatonin and jasmonate could not rescue the short root

224

or short hypocotyl phenotype compared with jasmonate alone (Fig. 7B, C). Given

10


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225

these independent results, we concluded that melatonin could not block jasmonate

226

signaling.

227 228

Melatonin Does Not Trigger Anthocyanin Catabolism.

229

Another possible explanation is that melatonin might trigger anthocyanin

230

catabolism when anthocyanin is over-accumulated. To test this possibility, we grew

231

pap1-D mutants in the presence of jasmonate to obtain high levels of anthocyanin

232

accumulation and then treated seedlings with melatonin at different time points before

233

determining their anthocyanin content. This showed that melatonin treatment could

234

not curtail the already accumulated anthocyanin even after 24 h treatment (Fig. 8),

235

indicating that melatonin does not elicit anthocyanin catabolism.

236 237

Melatonin Might Act on the Anthocyanin Biosynthesis Pathway.

238

To further dissect how melatonin antagonizes jasmonate-induced anthocyanin

239

production, we next determined the expression levels of anthocyanin biosynthesis

240

genes. As discussed, the anthocyanin biosynthesis pathway has been well-established

241

(Fig. 9A). We noticed here that jasmonate-induced the transcription of almost all the

242

key anthocyanin biosynthesis genes, but that melatonin treatment could not reverse

243

these inductions (Fig. S3).

244

These results suggest that melatonin does not cause repression of genes

245

associated with anthocyanin biosynthesis-related. Thus, we speculate that melatonin

246

might directly act on the enzymatic reactions to inhibit the biochemical steps

247

occurring during anthocyanin biosynthesis. Although mass spectrometry (MS) is an

248

ideal approach for quantifying the anthocyanin biosynthesis precursors, these

249

precursors are difficult to precisely measure because of their very low abundance and 11



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250

unstable nature. We, therefore, used an indirect method to identify the chemical step

251

that might be affected by melatonin. We tested all the commercial anthocyanin

252

biosynthesis precursors one by one with melatonin and jasmonate simultaneously to

253

determine which could rescue the repression effect caused by melatonin. It may be

254

that melatonin blocks production of the identified precursor to inhibit anthocyanin

255

biosynthesis. We first supplied the L-phenylalanine (L-Phe) that is the initial

256

precursor for anthocyanin biosynthesis. At concentrations as low as 5 µM L-Phe was

257

able to further boost anthocyanin biosynthesis in the presence of jasmonate (Fig. 9B),

258

however, co-treatment of L-Phe with jasmonate and melatonin could not suppress the

259

negative effects caused by melatonin unless an extremely high concentration of L-Phe

260

(100 µM) was supplied (Fig. 9B). We assume that extremely high concentration of

261

anthocyanin precursor (L-Phe) supply might overwhelm the melatonin effect. Then

262

we sequentially included cinnamate (cinnamic acid), naringenin chalcone, naringenin,

263

or dihydrokaempferol under a relatively low concentration (20 µM) and found that

264

only dihydrokaempferol could significantly rescue the melatonin effect (Fig. 9C).

265

Taken together, these results suggest that melatonin does not directly affect the

266

transcription level of the anthocyanin biosynthesis genes but might act on the

267

F3H-catalyzed dihydrokaempferol formation step.

268 269

DISCUSSION

270

Melatonin was initially identified in mammalian cells and was then reported to

271

also exist in plant cells. Although it has been reported that melatonin modulates a

272

plethora of physiological events in plants, its role in plant metabolism is not clear. In

273

this study, we first examined the direct role of melatonin in anthocyanin metabolism.

274

We included a wide range of melatonin concentrations and our results showed that 12


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275

melatonin may have a negative role in anthocyanin biosynthesis (Fig. 1). However,

276

anthocyanin biosynthesis capacity in Arabidopsis thaliana is relatively low under

277

normal growth condition that might limit these observations.

278

We then used jasmonate, which is an inducer of anthocyanin biosynthesis, to

279

examine whether melatonin modulates this induction response. In the presence of

280

melatonin, the induction effect by jasmonate is largely attenuated (Fig. 2 and Fig. S1).

281

In addition to exogenous melatonin treatment, we also tested the effect of melatonin

282

in transgenic plants over-producing melatonin and found that increasing endogenous

283

melatonin was sufficient to suppress jasmonate-induced anthocyanin production (Fig.

284

4).

285

There

are

two

possible

explanations

for

this

melatonin-mediated

286

antagonization of jasmonate-induced anthocyanin biosynthesis. One is that melatonin

287

abrogates jasmonate signal transduction and the other is that melatonin acts on the

288

anthocyanin biosynthesis route but not the jasmonate pathway. We first examined

289

jasmonate

290

jasmonate-responsive

291

physiological functions. All these characteristic jasmonate signaling features are not

292

changed by melatonin (Fig. 5–7). Furthermore, melatonin could also inhibit

293

jasmonate-induced anthocyanin accumulation in either jazQ or jasmonate biosynthesis

294

mutants (Fig. 3 and Fig. S2). Taken together, these results demonstrate that melatonin

295

does not antagonize jasmonate-stimulated anthocyanin production by abrogating

296

jasmonate signaling. This, therefore, excluded the first possible explanation.

signaling

activities marker

gene

by

observing

expression,

JAZ and

protein

other

stabilities,

jasmonate-related

297

Next, we tested the second possible explanation. We initially examined the

298

anthocyanin catabolism and transcription of genes encoding key enzymes in the

299

anthocyanin biosynthesis pathway. Melatonin does not affect anthocyanin catabolism 13



Page 14 of 32

300

even in jasmonate-treated pap1-D mutants where the anthocyanin levels were very

301

high (Fig. 8). Jasmonate treatment induced the expression of almost all the

302

anthocyanin biosynthesis-related genes but melatonin did not antagonize these

303

inductions (Fig. S3). Nevertheless, in our further metabolite supply assay, we showed

304

that the production of dihydrokaempferol might be suppressed by melatonin. We

305

tested individual anthocyanin biosynthesis precursors with both jasmonate and

306

melatonin and found that all the precursors ahead of dihydrokaempferol biosynthesis

307

could not suppress the inhibition effect of melatonin (Fig. 9). This indirect evidence

308

suggests that melatonin might directly inhibit the enzymatic activity of F3H that

309

catalyzes the formation of dihydrokaempferol. Future enzymatic analysis and protein

310

structure-based studies will be performed to determine the exact inhibition

311

mechanisms.

312

In conclusion, we reveal a negative role of melatonin in plant anthocyanin

313

metabolism, particularly during jasmonate-induced anthocyanin biosynthesis. We also

314

determined that melatonin does not abrogate jasmonate signaling and propose that

315

melatonin acts instead on the metabolism pathway, most likely at the F3H-catalyzed

316

dihydrokaempferol step. More interestingly, since many other environmental cues

317

could also induce anthocyanin production and melatonin acts in the anthocyanin

318

metabolism pathway, we assume that melatonin might be a crucial signaling molecule

319

for integrating multiple environmental and endogenous stimuli pathways and could be

320

a useful candidate for modulating anthocyanin production in fruits and vegetables.

321 322

AUTHOR INFORMATION

323

Corresponding Author

324

*E-mail: [email protected]; Tel: 86-25-85891837. 14


Page 15 of 32


325

Author Contributions

326

Y.A. performed experiments. Z.Z. conceived the study, analyzed data, and wrote the

327

manuscript.

328

Funding

329

This work is supported by the Fok Ying Tung Education Foundation (161023), the

330

Priority Academic Program Development of Jiangsu Higher Education Institutions

331

and Qing Lan Project to Z.Z.

332

Notes

333

The authors declare no competing financial interest.

334 335

ACKNOWLEDGMENT

336

We would like to thank Drs. Yangdong Guo and Wenbiao Shen for providing us with

337

materials and helpful discussions. We also thank the European Arabidopsis Stock

338

Centre for pap1-D mutants. We thank Dr. Emma Tacken for editing the English text

339

of this manuscript.

340 341

SUPPORTING INFORMATION

342

Supplemental Figure 1: Anthocyanin content of the wild-type (Col-0) seedlings grown

343

under long-day condition.

344

Supplemental Figure 2: Effects of exogenous melatonin on anthocyanin content of

345

jasmonate-deficient mutants.

346

Supplemental Figure 3: Gene expression analysis of anthocyanin biosynthesis related

347

genes.

348 349

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513 514 515 516

FIGURE CAPTIONS

517

Figure 1. Effects of exogenous melatonin on anthocyanin content of Arabidopsis

518

thaliana seedlings. 20


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519

Figure 2. Effects of exogenous melatonin on jasmonate-induced anthocyanin

520

accumulation.

521

Figure 3. Effects of exogenous melatonin on anthocyanin content of jazQ mutant

522

seedlings.

523

Figure 4. Effects of endogenous melatonin on anthocyanin content of Arabidopsis

524

seedlings.

525

Figure 5. Assays for determining JAZ1 protein stabilities.

526

Figure 6. Gene expression analysis.

527

Figure 7. Effects of exogenous melatonin on anthocyanin content and root or

528

hypocotyl growth phenotypes.

529

Figure 8. Effects of exogenous melatonin on anthocyanin catabolism.

530

Figure 9. Metabolic studies on the role of melatonin.

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FIGURES

Figure 1. Effects of exogenous melatonin on anthocyanin content of Arabidopsis thaliana seedlings. Representative images showing the anthocyanin accumulation levels in 10-day-old plants grown under continuous white light (A) or long-day condition (C) in the presence of different concentrations of melatonin. Quantitative quantification of anthocyanin contents in 10-day-old plants grown under either continuous white light (B) or long-day condition (D) in the presence of different concentrations of melatonin.

22


Page 23 of 32


For determining anthocyanin content in seedlings grown at long-day condition (C and D), samples were collected at zeitgeber time 8 (ZT8). Results are presented as means ± SD of three independent replicates. Bars marked with different letters differ significantly (Tukey post hoc test, P ≤ 0.05).

Figure 2. Effects of exogenous melatonin on jasmonate-induced anthocyanin accumulation. (A) Anthocyanin content was determined in 10-day-old seedlings grown on MS (Control), 50 µM jasmonate (JA), or 50 µM jasmonate plus 100 µM melatonin (JA + MT). Results are presented as means ± SD of three independent replicates. Bars marked with different letters differ significantly (Tukey post hoc test, p ≤ 0.05). (B) Representative images showing the different anthocyanin accumulation levels in 10-day-old plants grown on MS (Control), 100 µM melatonin (MT), 50 µM jasmonate (JA), or 50 µM jasmonate plus 100 µM melatonin (JA + MT).

23



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Figure 3. Effects of exogenous melatonin on anthocyanin content of jazQ mutant seedlings. (A) Representative images showing the different anthocyanin accumulation levels in 10-day-old plants grown on MS (Control), 50 µM jasmonate (JA), or 50 µM jasmonate plus 100 µM melatonin (JA + MT). (B) Anthocyanin content was determined from 10-day-old seedlings grown on MS (Control), 50 µM jasmonate (JA), or 50 µM jasmonate plus 100 µM melatonin (JA + MT). Results are presented as means ± SD of three independent replicates. Bars marked with different letters differ significantly (Tukey post hoc test, P ≤ 0.05).

24


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Figure 4. Effects of endogenous melatonin on anthocyanin content of Arabidopsis seedlings. (A) Representative images showing the different anthocyanin accumulation levels in 10-day-old plants grown on MS (Control) or 50 µM jasmonate (JA). (B) Anthocyanin content was determined from 10-day-old seedlings grown on MS (Control) or 50 µM jasmonate (JA). Results are presented as means ± SD of three independent replicates. Bars marked with different letters differ significantly (Tukey post hoc test, P ≤ 0.05).

25



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Figure 5. Assays for determining JAZ1 protein stabilities. (A) GUS activity was determined from 7-day-old (35S:JAZ1-GUS) seedlings treated with 50 µM jasmonate (JA), 100 µM melatonin (MT), or 50 µM jasmonate plus 100 µM melatonin (JA + MT) for 30 min. The same concentration of ethanol was used as a control treatment. Col-0 seedlings were used as negative controls. Results are presented as means ± SD of six independent replicates. Bars marked with different letters differ significantly (Tukey post hoc test, P ≤ 0.05). (B) Representative GUS staining images showing the JAZ1-GUS stabilities. Seven-day-old seedlings (35S:JAZ1-GUS) were treated with the same concentrations of reagents as shown in (A) for different time points. (C) Western blot analysis was performed to determine JAZ1-GUS protein levels. Seven-day-old seedlings (35S:JAZ1-GUS) were treated with the same concentrations of reagents as shown in (A) for 30 and 60 min.

26


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Figure 6. Gene expression analysis. qRT-PCR results showing jasmonate-inducible gene expressions. Seven-day-old Col-0 seedlings were treated with water (Control), 50 µM jasmonate (JA), or 50 µM jasmonate plus 100 µM melatonin (JA + MT) for 1 h. Results are presented as means ± SD of three independent replicates. Bars marked with different letters differ significantly (Tukey post hoc test, P ≤ 0.05).

27



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Figure 7. Effects of exogenous melatonin on anthocyanin content and root or hypocotyl growth phenotypes. (A) Anthocyanin content was determined from 10-day-old seedlings grown on MS (Control); 50, 100, or 200 µM melatonin (MT); 50 µM jasmonate (JA); or 50 µM jasmonate plus 50, 100, or 200 µM melatonin (JA + MT). (B) Root lengths of 7-day-old seedlings grown on MS (Control); 50, 100, or 200 µM melatonin (MT); 50 µM jasmonate (JA); or 50 µM jasmonate plus 50, 100, or 200 µM melatonin (JA + MT) vertically in light. (C) Hypocotyl lengths of seven-day-old seedlings grown on MS (Control); 50, 100, or 200 µM melatonin (MT); 50 µM jasmonate (JA); or 50 µM jasmonate plus 50, 100, or 200 µM melatonin (JA + MT) vertically in darkness. Results are presented as means ± SD of three independent replicates. Bars marked with different letters differ significantly (Tukey post hoc test, P ≤ 0.05).

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Figure 8. Effects of exogenous melatonin on anthocyanin catabolism. Anthocyanin content was determined from seedlings grown in the presence of 50 µM jasmonate for 7 d and then treated with water (Control) or 100 µM melatonin (MT) for different time periods. Results are presented as means ± SD of three independent replicates. Bars marked with different letters differ significantly (Tukey post hoc test, P ≤ 0.05).

29



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Figure 9. Metabolic studies on the role of melatonin. (A) Diagram of the anthocyanin biosynthetic pathway in Arabidopsis. Anthocyanin biosynthetic enzymes are shown in the diagram such as phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumaroyl: CoA-ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid

3’-hydroxylase

leucoanthocyanidin

(F3’H),

dioxygenase

dihydroflavonol (LDOX),

and

reductase

(DFR),

UDP-Glc:flavonoid

3-O-glucosyltransferase (UF3GT).

30


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(B) Anthocyanin content was determined from 10-day-old seedlings grown on MS (Control), 50 µM jasmonate (JA), 50 µM jasmonate (JA) plus different concentrations of L-phenylalanine (L-Phe), 50 µM jasmonate plus 100 µM melatonin (JA + MT), or JA + MT plus different concentrations of L-Phe. (C) Anthocyanin content was determined from 10-day-old seedlings grown on MS (Control), 50 µM jasmonate (JA), 50 µM jasmonate plus 100 µM melatonin (JA + MT), JA + MT plus 20 µM cinnamic acid, JA + MT plus 20 µM naringenin chalcone, JA + MT plus 20 µM naringenin, or JA + MT plus 20 µM dihydrokaempferol. Results are presented as means ± SD of three independent replicates. Bars marked with different letters differ significantly (Tukey post hoc test, P ≤ 0.05).

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Table of Contents Graphic

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Melatonin Antagonizes Jasmonate-Triggered Anthocyanin

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