1

Jasmonic acid facilitates flower opening and floral organ development

2

through the upregulated expression of SlMYB21 transcription factor

3

in tomato

4
5

Tomoko Niwa1, Takamasa Suzuki2, Yumiko Takebayashi3, Rie Ishiguro1,

6

Tetsuya Higashiyama4,5, Hitoshi Sakakibara1,3 and Sumie Ishiguro1,*

7
8

1

Graduate School of Bio-agricultural Sciences, Nagoya University, Nagoya, Japan;

92College of Bioscience and Biotechnology, Chubu University, Kasugai, Japan;

10

3

RIKEN Center for Sustainable Resource Science, Yokohama, Japan;

114Graduate School of Science, Nagoya University, Nagoya, Japan;

125Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya,

13

Japan.

14*Corresponding author

15
16

Self-archived accepted version (accepted on Dec. 11, 2017).

17This is a postprint version of an article published in Bioscience, Biotechnology, and18

Biochemistry Vol. 82, No. 2, pp. 292-303, 2018. Final version is available online at:19

www.tandfonline.com/Article DOI; 10.1080/09168451.2017.1422107.

20
21

CONTACT

22

Sumie Ishiguro (guronyan@@agr.nagoya-u.ac.jp)

23

Graduate School of Bio-agricultural Sciences, Nagoya University,

24

Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.

25

Phone: +81-52-789-4097

1

26

Abstract

27

Plants coordinate the timing of flower opening with pollen and gynoecium

28

maturation to achieve successful pollination. However, little is known about how the29

coordination is executed. We found that flower bud development was paused

30

immediately before flower opening in a jasmonic acid (JA)-insensitive tomato mutant,31

jai1-1. Phytohormone measurement and RNA analysis in flower buds revealed that32

newly synthesized JA peaked at two days before flower opening and the expression of a33

transcription factor gene SlMYB21 delayed in jai1-1. Buds of transgenic tomato plants

34

expressing an artificial repressor, AtMYB24-SRDX, which was expected to impede the35function of SlMYB21, aborted flower opening and resembled those of jai1-1.

36Furthermore, the AtMYB24-SRDX plants produced abnormal pollen grains deficient in37

germination and pistils that did not support pollen tube elongation. We concluded that38

JA facilitates the expression of SlMYB21, which coordinates flower opening, pollen

39

maturation, and gynoecium function in tomato.

40

41

Keywords: flower opening; jasmonic acid; male and female fertility; SlMYB21

42transcription factor; Solanum lycoperisicum

43
44
45

Short title

46

JA coordinates fertility with flower opening through SlMYB21

47
48
49

2

50

Introduction

51
52

The flower is the most important reproductive organ in angiosperms. The flowers

53

release mature pollen grains from anthers, attract pollinators by color and scent, attach54

the pollen grains to the pollinators, and accept pollen grains at stigmas. Therefore,55

coordination of flower opening with pollen maturation and gynoecium maturation is56

important for successful pollination and subsequent fertilization. Flower opening is57

regulated by both the internal environment such as pollen and ovule development and58

circadian rhythms and the external environment such as light, temperature, and59

humidity.1, 2) Moreover, many phytohormones, such as auxin, gibberellin (GA), abscisic

60

acid (ABA), ethylene, and jasmonic acid (JA), have been reported to be involved in the61

regulation of flower opening with relation to both internal and external cues.1)

62Involvement of JA in the regulation of flower opening was initially reported in the63

analysis of a JA-deficient mutant, defective in anther dehiscence1 (dad1), in

64

Arabidopsis.3) The dad1 flower buds developed normally until the end of the middle

65

stage, which corresponded to two days before flower opening in the wild type (WT). In66

the later stage, however, the development of the mutant flower buds was retarded in67

comparison to WT until flower opening, and as a consequence, more unopened buds

68

were clustered in the inflorescence of dad1.3, 4) The dad1 flowers also showed delayed

69

anther dehiscence and pollen grain infertility, and all three defects including delayed70

flower opening were rescued by exogenous application of JA.3) Similar phenotypes

71

appeared in other JA biosynthesis mutants such as allene oxide synthase,72

oxophytodienoate reductase3 (opr3), double mutants lipoxygenase3 lipoxygenase4,5-8)

73

and a JA-insensitive mutant, coronatine insensitive1 (coi1).9) COI1 is a JA receptor

74

essential for all the JA responses including defense and stress responses in

75

Arabidopsis.10) COI1 is an F-box protein that forms a complex with a jasmonate3

76

ZIM-domain protein, and the complex specifically binds to jasmonoyl-L-isoleucine

77(JA-Ile). Hence, JA-Ile is thought to be an active form of JA.11, 12) These observations

78

indicate that JA is an activator of flower opening, anther dehiscence, and pollen

79

development in Arabidopsis.

80

Regulation of flower opening by JA was also reported in other plants. In Brassica

81

rapa, flower opening was suppressed in the transformants expressing an antisense gene82

of BrDAD1, which is the putative ortholog of Arabidopsis DAD1, and the phenotype83

was restored by application of JA.13) In wild tobacco, Nicotiana attenuata, a series of84

transgenic plants in which JA biosynthetic genes such as ALLENE OXIDE CYCLASE

85(AOC), or a JA perception gene, COI1, were silenced showed delayed flower opening or

86

incomplete corolla expansion phenotypes.14) In rice, opening of florets was induced by87

treatment of methyl jasmonate, and the floret opening as well as anther dehiscence was88

impaired in the dysfunctional mutants of OsJAR1, which encodes an enzyme forming

89

JA-Ile.15, 16) Enhanced flower opening by methyl jasmonate treatment was also observed

90

in Brassica napus.17)

91

Along with the defects in flower opening, JA-related mutants often affect the92

development of floral organs. Rice mutant extra glume 1 (eg1), which is a mutation in a93homolog of DAD1, changed floral organ identity and number.18) Tomato mutant

94

jasmonic-acid insensitive1 (jai1), of which the causal gene is SlCOI1, is a unique95

ortholog of Arabidopsis COI1 and showed reduced pollen viability and female

96

sterility.19, 20) Dobritzsch et al. proposed that the reduced pollen fertility in jai1 is

97

attributed to the premature tapetum degradation and premature anther dehiscence in the98

absence of normal JA signaling.21) These observations revealed that JA is not only a99

common regulator of flower opening in many plants but also involved in the100

development of floral organs including male and female gametophytes in a

101

species-dependent manner. However, their mechanisms are largely unknown.
4

102

A transcriptome analysis comparing JA-treated and not-treated stamens of103

Arabidopsis opr3 mutants revealed that two R2R3-type MYB transcription factors,

104

AtMYB21 and AtMYB24, were strongly induced by JA.22) T-DNA insertion mutants of105

atmyb21 exhibited delayed flower opening, shorter anther filaments, delayed anther106

dehiscence, and reduced male fertility, resembling the phenotype of JA-deficient107

mutants.22) Another transcriptome analysis identified three genes AtMYB21, AtMYB24,

108

and AtMYB57 as GA-dependent genes in flower buds.23) All atmyb21 phenotypes were109

enhanced in atmyb21 atmyb24 double mutants and were further exacerbated in atmyb21

110

atmyb24 atmyb57 triple mutants, suggesting overlapping functions of these three

111

R2R3-MYB genes.22, 24) The authors concluded that GA acts through JA to control

112

expression of the R2R3-MYB genes and promote stamen filament elongation.23)
Involvement of putative orthologs of AtMYB21/24/57 in flower opening has also

113
114

been

115

Pro35S:AtMYB24-SRDX gene, which consisted of the cauliflower mosaic virus 35S

116

promoter (Pro35S) and the AtMYB24 coding sequence fused to the sequence of an EAR

117

motif repression domain (SRDX), exhibited a deficiency in flower opening.25) The EAR

118

motif

119

ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR in Arabidopsis and has

120

the ability to change transcription activators to repressors when it is fused to the121

activators.26, 27) In petunia and Nicotiana attenuata, suppression of the EMISSION OF122

BENZENOIDS II (EOBII) gene, a close homolog of AtMYB21/24, by RNA interference

123

caused a failure of flower opening.28) These findings indicate that the ortholog(s) of124

AtMYB21/24/57 regulate flower opening in diverse plant species. However, the125

relationship between JA signaling and regulation of AtMYB21/24/57 ortholog(s) has not126

yet been elucidated in plants other than Arabidopsis.

127

reported

was

in

other

originallyplants.

identified

Transgenic

asatoreniatranscription

plantsexpressing

repression

domaintheofIn this study, we found that tomato plants having jai1-1, a strong allele of jai15

128

mutation, showed delayed flower opening. Phytohormone measurement and transcript129

analysis revealed that JA stimulated the expression of the SlMYB21 gene, a unique130

ortholog of AtMYB21/24/57 in tomato. We showed that transgenic tomato expressing a

131chimeric transcription repressor for SlMYB21 exhibited a more exacerbate phenotype

132

than jai1-1 particularly in male and female sterility. We will discuss the involvement of133

JA signaling and SlMYB21 in the regulation of flower opening and floral organ

134

development in tomato.

6

135

Materials and methods

136

Plant materials and growth conditions.

137

Tomato cultivar Micro-Tom

138(Solanum lycopersicum L. cv. Micro-Tom) was used unless otherwise indicated. jai1-1,

139

a deletion allele of the SlCOI1 gene (Solyc05g052620) in a Micro-Tom background,140

was described previously.19) Due to the strong sterility of jai1-1 homozygotes, jai1-1

141

was maintained as a heterozygous line, and homozygous jai1-1 plants were identified

142

by a PCR-based genotyping.19) We established a WT line having a homozygous

143

unmutated SlCOI1 allele from the progeny of a jai1-1 heterozygote and named it

144“MT-GH”. MT-GH was used as the WT control in most experiments in this study. For

145

RNA sequencing (RNA-seq), a line of Micro-Tom (MT-J, TOMJPF00001) obtained

146

from the National Bio-Resource Project (http://tomato.nbrp.jp/indexEn.html) was used.

147

Another cultivar of tomato (cv. Momotaro 8, Takii, Japan) was also used for some148

experiments. Seeds were sown on pre-fertilized soil (Nippi No. 1, Nippon Hiryo, Japan)

149

and grown under fluorescent white light (170 µmol/s/m2) of 16 h light and 8h darkness

150

at 25°C.

151

RNA preparation and gene expression analysis.

152

A whole flower bud or the153

floral organs of a bud were harvested and immediately frozen in liquid nitrogen. Total

154

RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN, www.qiagen.com).

155

Extracted RNA was reverse transcribed with ReverTra Ace qPCR RT Master Mix with

156

gDNA Remover (Toyobo, www.toyobo-global.com). qRT-PCR was performed using

157

the

158

www.thermofisher.com) and THUNDERBIRD SYBR qPCR Mix (Toyobo). RNA

159

yields and reverse transcription efficiencies were normalized by the amount of DNAJ (a160

subset of tomato DnaJ/Hsp40 homologs comprising Solyc04g009770, Solyc05g055160,

StepOneReal-Time

PCR

system

7(ThermoFisherScientific,

161

and Solyc11g006460) mRNA or SAND (Solyc03g115810) mRNA, as described in the162

literature.29) The primers used in qRT-PCR are listed in Supplemental Table S1.

163

RNA-seq analysis was carried out as described previously.30) Reference cDNA

164

sequences were extracted from a whole genome sequence database (version SL2.40 of

165

Solanum lycopersicum cv. Heinz 1706) by use of ITAG2.3 gene prediction information

166(Sol Genomics Network, solgenomics.net).

167
168

Measurement of phytohormones.

Each flower bud was separately harvested

169

from an inflorescence and immediately frozen in liquid nitrogen. Extraction and170

purification

171

Quantification of phytohormone species excepting JA-Ile were extracted and measured

172

as described previously.31, 32) For JA-Ile quantification, stable isotope-labeled JA-Ile

173([2H2]N-[(-)-Jasmonyl]-isoleucine, OlChemim, www.olchemim.cz/) was used as the174

internal standard. Detection was performed by ultra-high performance liquid

175

chromatography (UHPLC)-electrospray interface (ESI) and quadrupole-orbitrap mass

176

spectrometer (UHPLC/Q-Exactive™; Thermo Fisher Scientific) with an AQUITY

177

UPLC HSS T3 column (2.1 × 100 mm; 1.8 µm, Waters).

ofphytohormones

wereperformed

asdescribed

previously.31)

178
179

Construction of plasmids and transgenic plants.

To construct the plasmid

180

pBCKK-Pro35S:AtMYB24:SRDX expressing the AtMYB24-SRDX gene under the181

control of the cauliflower mosaic virus 35S promoter, the Pro35S:MYB24-SRDX

182

fragment was transferred from a plasmid 35S:MYB24-SRDX/pEntry, which is a183

previously reported entry plasmid constructed on p35SSRDXG vector,33) to the pBCKK

184

binary vector 34) by a Gateway LR reaction (Thermo Fisher Scientific). Transformation

185of tomato plants was performed as described previously.35)

186
8

187

Microscopy.

An

SZX12

stereomicroscope(Olympus,

188

www.olympus-global.com/en/) was used for observation of the morphologies of flower

189

buds and flowers. To observe pollen tube growth in vivo, manually pollinated pistils

190

were harvested 24 h after pollination, fixed in ethanol:acetic acid (3:1) for 24 h, and191

treated for 12 h with 5N NaOH. After being rinsed three times in water, the pistils were192

stained in 0.1 % aniline blue dye solution in 0.1 M K-phosphate buffer (pH 11) for 1 to

193

4 h. The pistils were mounted on a slide with 80% (v/v) glycerol solution and observed

194

by a fluorescence microscope BX60 (Olympus) under UV light excitation (Filter set

195

WU, excitation 330-385 nm, emission >420 nm). To visualize the pollen nuclei, pollen

196

grains in dehisced anthers were suspended in 1 µg/ml 4',6-diamidino-2-phenylindole

197(DAPI) solution in 1 x PBS (130 mM NaCl, 5.1 mM Na2HPO4, 1.6 mM KH2PO4) by

198

brief vortexing. The pollen suspension was mounted on a slide and observed by

199

fluorescence microscopy with UV excitation. To observe the female gametophytes,

200

ovules were collected from newly opening flowers, fixed in ethanol:acetic acid (9:1 v/v)

201

solution for 2 h, and cleared in a modified Hoyer’s solution (7.5 g gum arabic, 100 g

202

chloral hydrate, 5 ml glycerol, 30 ml H2O). Specimens were observed by differential

203

interphase contrast microscopy.

9

204

Results

205
206

Delayed flower opening in a JA-insensitive mutant

207

In our growth condition, most of the WT Micro-Tom plants produced nine208

flowers in the primary inflorescence. Fig. 1A shows a typical primary inflorescence in

209

which the first (a) and second (b) flowers have opened and the next three buds (c to e)210

have petals longer than the sepals (hereafter called the petal-emerged phase). The other211

four buds (f to i) did not show their petals (Fig. 1A). We observed the daily212

developmental process of each flower bud in the inflorescence and found that one

213

flower newly opened every day in basal to apical order, and once-opened flowers

214

continued to open for two days before senescence. The duration of the petal-emerged

215

phase (including the partially opened bud) was three days on average (Fig. 1D, F).

216

To analyze the roles of JA on the bud development and flower opening in tomato,217

we observed a JA-insensitive tomato mutant, jai1-1, which is a deletion allele of jai1

218

lacking a whole exon 2 and exon 3 of the SlCOI1 gene.19, 20) Fig. 1B shows a primary219

inflorescence of jai1-1 at the same developmental stage as that of the WT inflorescence

220

shown in Fig. 1A. The size and shape of the four youngest flower buds (f to i) and the221

next three petal-emerged buds (c to e) of jai1-1 were similar to those of the222

corresponding buds in WT. However, the two largest buds (a and b) in jai1-1 had not223

yet opened, whereas the counterparts in WT had fully opened (Fig. 1A, B). Fig. 1C

224

shows another inflorescence of jai1-1 when the first two flowers (a and b) were partly

225(but not yet fully) opened. All the other buds showed the petals. Continuous observation

226of the inflorescence revealed that most of the jai1-1 flower buds required four to five

227

days for passing though the petal-emerged phase and an additional two days for the228

partially opened phase before complete opening (Fig. 1E, F). These results indicated229that the jai1-1 flower buds paused the development for three days in comparison to WT
10

230

before opening. Hence, we concluded that JA has an important role in facilitating flower

231

opening in tomato.

232
233

Levels of phytohormones in developing WT and jai1-1 flower buds

234

The above observations prompted us to examine whether JA and other235

phytohormones are biosynthesized and accumulated in tomato flower buds prior to236

flower opening and whether the levels might be different between in WT and jai1-1

237

flower buds. To describe the flower development of Micro-Tom, we used the bud age in

238

days. We defined the age of the flower bud that attained 4 mm in length as a “day-1 bud”

239

and larger flower buds on the following days as a “day-X bud” (Fig. 2A). Typically, the240

day-2 bud became longer than 5 mm but its calyx was still closed. On day 3, the calyx

241

opened and petals could be observed. On day 4, petals became longer than the sepals.

242

On day 5, petals enlarged, whereas petal color remained pale green. On day 6, petal

243

color changed to light yellow. On day 7, petals began to elongate outward, which meant244

initiation of flower opening. On day 8, petals elongated completely and bent outward.

245

The flower fully opened, and this morphology continued to the end of day 9, except

246

petals partially closed during the night. Then, flower began to close on day 10 and was247

senesced on day 11. The size and morphology of jai1-1 flower buds were equivalent to248

those in WT until day 5, but the development was delayed thereafter (Fig. 2B). The

249

day-6 bud of jai1-1 was indistinguishable from that on day 5. Petals began to turn250

yellow on day 7, but they were still tinged with green and never opened on day 8. The

251

bud finally began to open on day 9 and was fully open on day 10. Petals closed on day

252

12 (Fig. 2B). Consistent with the observation shown in Fig. 1, bud development in253

jai1-1 was retarded for two to three days at the petal-emerged phase, and subsequent

254

flower opening and closing proceeded two days later than in WT.

255

We performed simultaneous measurement of phytohormones from the day-1 bud
11

256

to flower opening. Three flower buds were harvested for each stage (day 1 to day 7 for257

WT and day 1 to day 9 for jai1-1), and an individual bud was separately used for258

hormone extraction and measurement. It is an important advantage of this method that259

the levels of various hormones in the same samples can be compared directly. First, we260

analyzed JA and its biologically active form, JA-Ile, in WT. Both molecules began to261

increase on day 3, reached a sharp peak on day 5, at which petal growth was nearly262

completed but the color had not yet turned yellow, and decreased to the basal level263

when the flower began opening on day 7 (Fig. 2A, C). In contrast, accumulation of JA

264

and JA-Ile in jai1-1 was hardly detectable throughout flower bud development (Fig. 2B,

265C), which confirmed a previously reported result.21) These results suggested that the JA

266

and JA-Ile accumulated in the day-5 buds is important for facilitating the subsequent

267

flower opening.

268

Similar peak formation around day 5 was observed for the levels of269

indole-3-acetic acid (IAA) and abscisic acid (ABA), whereas gibberellin A1 (GA1)270

reached the peak slightly earlier at day 3 (Fig. 2C). However, there were only small271

differences between WT and jai1-1. Two cytokinins, N6-(Δ2-isopentenyl) adenine (iP),272

and trans-zeatin (tZ), temporary increased in the day-2 flower buds of both WT and273

jai1-1 (Fig. 2C). Intriguingly, tZ increased in the day-7 bud in WT and day-9 bud in274

jai1-1, both of which corresponded to opening flowers. Although the difference on day

275

7 was not statistically significant due to large deviations in WT, all three measured

276

values for WT were larger than those for jai1-1. Hence, it indicated that the level of tZ

277

might be causally related to flower opening.

278

Salicylic acid (SA) increased gradually during bud development but not279

differentially between WT and jai1-1, suggesting no relation to flower opening (Fig.

280

2C). GA4 was possibly accumulated in young flower buds and the opening flower in

281

WT, though the reliability of this finding is low due to large deviations among samples
12

282

(Fig. 2C). In summary, simultaneous phytohormone measurement revealed that the283

levels of JA, JA-Ile, and tZ were affected by jai1-1 mutation during flower bud

284

development. In the following experiments, we focused on the expression of285

biosynthesis and downstream genes of JA.

286
287

Expression of JA-related genes during flower bud development in WT and jai1-1

288

Among genes encoding JA biosynthetic enzymes, putative orthologs of289

Arabidopsis AOC (hereafter AtAOC) and OPR3 (AtOPR3) have been identified in290

tomato,36, 37) and we refer to them as SlAOC (Solyc02g085730) and SlOPR3

291(Solyc07g007870), respectively. The expression levels of SlAOC and SlOPR3 were292

measured by quantitative reverse transcription PCR (qRT-PCR). In WT, the SlAOC

293

expression was low until day 2, rapidly increased to the highest level at day 3, and294

gradually decreased by flower opening at day 7 (Fig. 3A). SlOPR3 was moderately

295

expressed throughout development with a broad peak around day 4 (Fig. 3B). It was

296

characteristic that these peaks slightly preceded (by one to two days) the accumulation297

of JA and JA-Ile. In contrast, the level of SlAOC expression in jai1-1 was kept low

298

throughout bud development and was significantly lower than that in WT during day 1299

to day 6 (Fig. 3A). SlOPR3 was expressed in the mutant at a level comparable to WT,

300

but the peak was unclear (Fig. 3B). The reduced expression of JA biosynthetic genes,301

especially of SlAOC, was consistent with the deficient accumulation of JA and JA-Ile in302

the mutant.

303

The weak correlation between SlOPR3 expression and JA accumulation prompted

304

us to examine whether AtOPR3 homologs other than SlOPR3 in the tomato genome

305

contribute to JA synthesis in developing flower buds. A BLAST search revealed six

306

genes as being similar to SlOPR3 in the tomato genome (Supplemental Fig. S1A) but no307

genes homologous to SlAOC. We analyzed an RNA-seq data set consisting of a series of
13

308

developing WT flower buds at days 1, 2, 3, 5, 6, and 7 and found that SlOPR3 as well as

309

SlAOC was highly expressed in unopened flower buds with a peak at day 3, whereas the310

expression

311

Solyc10g086220, were much lower than that of SlOPR3 (Supplemental Fig. S1,

312

Supplemental Table S2). Solyc10g086220 showed moderate expression throughout bud

313

development, but it has been shown that this gene, designated LeOPR1 or SlOPR1, is314

not involved in JA synthesis.38) Therefore, we concluded that SlOPR3 is predominantly

315

involved in JA synthesis in developing flower buds and the contribution of other

316

SlOPR3 homologs is small, if any.

levels

of

most

other

SlOPR3homologs

intomato,excepting

317

In Arabidopsis, three R2R3-MYB genes, AtMYB21, AtMYB24, and AtMYB57,

318

regulate flower opening and stamen elongation downstream of the JA signaling319

pathway.22, 23, 39) To identify the orthologs of these genes in tomato, we performed a320

similarity analysis of a subset of tomato and Arabidopsis R2R3-MYB genes.40) We found321

that only SlMYB21 (Solyc02g067760) belonged to the same clade as AtMYB21/24/57,

322

suggesting that SlMYB21 is a unique ortholog of AtMYB21/24/57 (Fig. 3D). The

323

expression of SlMYB21 was first detected in the day-3 bud, in which JA and JA-Ile

324

accumulation began, and reached the maximum level on day 6, which was one day later

325

than the peaks of JA and JA-Ile accumulation (Figs. 2A, C, 3C). The expression profile326

was consistent with the data of RNA-seq analysis (Supplemental Fig. S1D). The

327

RNA-seq analysis also revealed that the expression level of SlMYB21 in developing

328

flower buds was much higher than those of the other nine genes shown in Fig. 3D

329(Supplemental Fig. S1D, Supplemental Table S2). In jai1-1 buds, the first detection of330

SlMYB21 expression was delayed to day 5, which was two days later than the timing in

331

WT (Fig. 3C). The expression increased gradually and finally reached the maximum

332

level on day 9, which was comparable to the WT level at day 6 (Fig. 3C). The results

333

suggested that the expression of SlMYB21 does not require, but is facilitated by,
14

334

activated JA signaling in flower buds. The delayed flower opening in jai1-1 was335

coincident with the delayed expression of the SlMYB21 gene.

336
337

Suppression of SlMYB21 mimics the delayed flower opening phenotype of jai1-1

338

In order to test whether SlMYB21 is involved in flower opening in tomato, we339

constructed transformants of Micro-Tom containing the Pro35S:AtMYB24-SRDX gene,340

which expressed a chimeric repressor protein, AtMYB24-SRDX, under the control of341

the cauliflower mosaic virus 35S promoter.33) Because AtMYB24 is one of the closest

342

homologs of SlMYB21 (Fig. 2F), the expression of Pro35S:AtMYB24-SRDX is expected343

to repress the transcription of SlMYB21 target genes in tomato. Among independently

344

obtained 12 transformants redifferentiated from transgenic calli, six plants showed a345

remarkable phenotype. Flower buds of the transformants showed severely retarded346

opening or never opened until senescence (Fig. 4A, B). This phenotype resembles those347

of transgenic torenia plants containing the same gene 25) as well as of Arabidopsis

348

atmyb21 atmyb24 double mutants of strong alleles and atmyb21 atmyb24 atmyb57 triple

349

mutants.22, 23) Taken together with the uniqueness of SlMYB21 in the clade of350

AtMYB21/24/57 (Fig. 3D), we concluded that AtMYB24-SRDX acts as a repressor of351

SlMYB21 in tomato.

352

We compared the bud and flower development of the AtMYB24-SRDX

353

transformants to that of WT and jai1-1 in the period from day 3 to day 10 (Fig. 4C–E).

354

Until day 5, the bud morphologies of all three lines, in particular the size and color of

355

organs, were indistinguishable. The first difference was observed at day 6, when the356

WT petals began to turn yellow. Similar to jai1-1, the color of AtMYB24-SRDX petals

357

remained pale green at this stage (Fig. 4C–E). Flower buds of AtMYB24-SRDX plants358

were severely delayed in flower opening, and in extreme cases they never opened even359

when petals began to senesce on day 13 (Fig. 4E). Nevertheless, observation of the
15

360

inner organs revealed that the timings of anther color change (on the day 7) and anther361

dehiscence (on the day 8) were not delayed in either jai1-1 or AtMYB24-SRDX plants

362(Fig. 4C-E). These observations indicated that the SlMYB21 gene regulates the timing of363

flower opening but not of anther dehiscence in tomato.

364
365

Deficient male and female fertility in AtMYB24-SRDX flowers

366

Flowers of AtMYB24-SRDX plants occasionally bore fruits but they contained few

367

seeds (Fig. 5A), suggesting that SlMYB21 plays an important role in male and/or female

368

organs. A similar phenotype is observed in jai1-1 (Fig. 5A), and the sterility has been369

attributed to the deficiency in male and female organs, though the detailed mechanism

370

has not been elucidated.19, 20) qRT-PCR analysis revealed that the SlMYB21 gene was371

expressed in all floral organs of Micro-Tom flower buds immediately before flower

372

opening (day 6), and the levels in petals, anthers, filaments, and styles were more than373

two times higher than that in sepals (Fig. 5B). Thus, we attempted to examine how

374

SlMYB21 contributes to the fertility. We used transgenic AtMYB24-SRDX plants

375

generated in the background of commercial cultivar Momotaro 8, of which the large

376

organ size made dissection easier. Two out of 17 independent transformants of this377

cultivar showed the obvious opening-deficient phenotype in flowers (Supplemental Fig.

378

S2) and were used for further analysis.

379

We manually pollinated AtMYB24-SRDX pollen grains onto the WT stigma and380

tested pollen tube elongation by aniline blue staining. In contrast to the control

381

experiment, in which the WT pollen elongated many pollen tubes, a few pollen tubes

382

were observed in a style when AtMYB24-SRDX pollen was pollinated (Fig. 5C). The

383

result that only a small number of pollen grains remained on the stigma indicated that384

most of the pollinated pollen grains dropped out during the process of aniline blue

385

staining, presumably because they did not anchor to the stigma by their germinated
16

386

pollen tubes. Defective pollen tube elongation was also observed for jai1-1 pollen

387

grains. In this case, many pollen grains germinated but aborted their tube growth in the

388

styles (Fig. 5C). A morphological abnormality was also found in AtMYB24-SRDX

389

pollen grains. Fully developed pollen grains in tomato contain two nuclei, a large

390

vegetative nucleus and a small or elongated generative cell nucleus. But DAPI staining391

of pollen grains collected from dehisced anthers revealed that the generative nuclei in a392

considerable proportion (more than 10%) of AtMYB24-SRDX pollen grains had already393

divided into two putative sperm cell nuclei (Fig. 5D, E). Such generative cell division in

394

ungerminated pollen grains was rarely observed in WT and jai1-1 pollen grains (Fig.

395

5D, E), even though the flowers had senesced. Therefore, we concluded that SlMYB21

396

is required for the development and function of pollen grains.

397

To evaluate the female fertility of AtMYB24-SRDX flowers, we pollinated WT

398

pollen grains onto the AtMYB24-SRDX stigma and observed the pollen tube elongation.

399

Most pollen grains germinated, but the pollen tubes frequently terminated their growth

400

in the stigma or in the style, suggesting that SlMYB21 is required for stigmas and styles

401

to facilitate pollen tube elongation (Fig. 5F). Pistils of jai1-1 as well as WT did not402

show such defects. The failure of pollen tube elongation might be attributed to the403

developmental defects of ovules. However, we could not find any abnormalities among404

the ovules of AtMYB24-SRDX, jai1-1, and WT plants. In conclusion, SlMYB21 is405

necessary for the functional development of both male and female organs.

406

17

407

Discussion

408
409

In our growth conditions, Micro-Tom plants open a new flower every day in their410

primary inflorescences. This means that an inflorescence meristem of a primary shoot411

produces one flower bud every day, so that the inflorescence consists of a series of412

flower buds the ages of which differ by one day from the neighbors. We found that413

jai1-1 flower buds develop normally until day 5 of bud age, but subsequently delay the414

growth and require two to three extra days for opening. This phenotype resembles those415

of Arabidopsis JA-deficient and JA-insensitive mutants.3-9) In WT plants, JA and JA-Ile

416

peak on day 5. After day 5, the developmental delay of jai1-1 buds appears, suggesting

417

that activation of JA signaling is required to trigger the process of flower opening. The

418

accumulated JA and JA-Ile levels in WT flower buds were higher than those in419

wounded tomato leaves,41-43) suggesting that strong JA signaling is required for the420

flower opening. An RNA measurement showed that the level of SlAOC mRNA was

421

temporally increased in the day-3 buds, which preceded the peak of JA accumulation by422

two days. The induction of SlAOC is dependent on the function of SlCOI1, the causal

423

gene of jai1-1, which accounts for the failure of JA accumulation in jai1-1 buds.

424

Another JA biosynthetic gene, SlOPR3, was moderately expressed throughout bud

425

development but was also weakly upregulated in an SlCOI1-dependent manner. Hence,426

it seems likely that the activation of a positive feedback loop of JA synthesis is427

necessary for the promotion of flower opening. Activated JA signaling accelerates the428

expression of SlMYB21, the expression of which coincides with flower opening.

429

Furthermore, strong repression of SlMYB21 caused a failure of flower opening in

430

tomato, as reported in other plants such as torenia and petunia. These indicate that JA

431

facilitates flower opening through the activation of SlMYB21 expression.

432

With respect to the development of stamens in jai1-1 flower buds, anther color
18

433

change and anther dehiscence normally occurred, whereas petal elongation was delayed.

434This differential development in jai1-1 was previously explained as premature anther435

dehiscence and premature pollen release.21) However, based on our morphology and436

gene expression analyses, we proposed that anther wall development was normal but

437

petal expansion was delayed in jai1-1.

438

Simultaneous phytohormone measurement revealed that GA1 peaked at day 3 in439

parallel with the expression of SlAOC. In Arabidopsis, GA is required for the induction

440

of JA biosynthetic genes such as DAD1, and JA controls the expression of441

AtMYB21/24/57 genes and promotes stamen filament elongation.23) Auxin was also442

accumulated simultaneously with or slightly earlier than JA in the day-4 to day-5 flower443

buds. Arabidopsis AUXIN RESPONSE FACTOR6 (ARF6) and ARF8 are required for444

the activation of DAD1 expression,4) and also AtMYB21/24 expression, by both445

JA-dependent and independent pathways.39) Our results suggested the existence of a446

similar mechanism in tomato. We also found a temporal accumulation of cytokinins and447

abscisic acid during flower bud development. In particular, an increased tZ level in448

parallel with flower opening is characteristic, though their biological meanings have not449

yet been explained.

450

Observation of AtMYB24-SRDX flowers revealed that SlMYB21 is not only451

required for flower opening but also plays important roles in male and female organ

452

development. However, the phenotypic characteristics of AtMYB24-SRDX tomato were453

not identical to those of atmyb21 atmyb24 double mutants in Arabidopsis. In addition to454

the lack of petal expansion leading to unopened flowers, the atmyb21 atmyb24 double

455

mutants with a combination of knockout alleles showed short stamen filaments,456

undehisced anthers, and reduced pollen fertility, but female fertility was normal.22, 39) In

457

contrast, the AtMYB24-SRDX tomato showed abnormal pollen development, impaired458

pollen tube elongation, and deficient stigma/style function, but anther dehiscence was
19

459

normal. The effect on filament elongation cannot be evaluated due to innate short460

filaments in tomato. It is suggested that the functions of SlMYB21 and AtMYB21/24

461

have varied in the course of morphological diversification between tomato and462

Arabidopsis. This difference partly explains the phenotypic difference between

463

Arabidopsis coi1 and tomato jai1. The former shows a failure of filament elongation464

and anther dehiscence, which results in the production of dysfunctional pollen grains.9)465

In contrast, the latter shows no defect in anther dehiscence but shows abnormal male466

and female fertility.19, 20) We frequently found aborted pollen tube elongation of jai1-1

467

pollen grains, which is consistent with their reduced viability reported previously;19)468

however, we have not found any reason for female sterility.

469

We also found that there were considerable differences between jai1-1 mutants

470

and AtMYB24-SRDX plants in the phenotypes of male and female organs. Pollen grains

471

of AtMYB24-SRDX plants hardly germinated on the stigma, whereas jai1-1 pollen

472

grains germinated but aborted pollen tube elongation. Precocious division of generative473

cells in pollen grains frequently observed in AtMYB24-SRDX plants occurs only rarely474

in jai1-1 pollen grains. Furthermore, styles of AtMYB24-SRDX plants inhibit pollen tube

475

elongation, whereas jai1-1 styles do not show such a defect. These observations indicate

476

that the SlMYB21 gene is required for the functional development of both male and477

female organs after the opening of flowers. Relatively weak defects in jai1-1 mutants in

478

comparison to AtMYB24-SRDX plants might be explained by retarded but increased479

expression of SlMYB21 genes in jai1-1 flowers.

480

In summary, JA synthesized in developing flower buds accelerates the expression

481

of the SlMYB21 gene, which coordinately induces petal elongation and functional482

development of male and female organs in tomato. JA might determine the timing of483

flower opening by integrating the signals of other hormones such as GA and auxin.

484

Although the role of JA in inducing the expression of AtMYB21/24 or SlMYB21 genes is
20

485

common between Arabidopsis and tomato, the functions of these MYB genes have486

diverged between the two species.

21

487

Author contributions

488

T.N., T.S., T.H., H.S., and S.I. designed and coordinated the experiments. T.N.,

489

T.S., Y.T., and R.I. carried out the experiments and analyzed the results. T.N. and S.I.

490

wrote the manuscript. All the authors have read and approved the final manuscript.

491
492

Acknowledgements

493

We thank Ms. Tomomi Shinagawa and Mr. Naoki Takahashi (Nagoya University)494

for her/his skilled technical assistance, Dr. Shunsuke Imanishi (National Agriculture495

and Food Research Organization) and Professor Mikio Nakazono (Nagoya University)496

for helpful discussion, Professor Kenji Matsui (Yamaguchi University) for technical497

advice, Professor Gregg A. Howe (Michigan State University) for providing the jai1-1

498

seeds, and Dr. Nobutaka Mitsuda (National Institute of Advanced Industrial Science and499

Technology) for providing 35S:MYB24-SRDX/pEntry and pBCKK plasmids.

500

Micro-Tom seeds (TOMJPF00001) were provided by University of Tsukuba, Gene

501

Research Center, through the National Bio-Resource Project of the AMED, Japan.

502

Homology alignment was performed with Clustal W provided at the DDBJ website.

503
504
505

Disclosure statement
No potential conflict of interest was reported by the authors.

506
507

Funding508This work was supported by the Cabinet Office, Government of Japan, under the509

SIP Program "Technologies for creating next-generation agriculture, forestry and510

fisheries" (funding agency: Bio-oriented Technology Research Advancement Institution,

511

NARO, Japan) to S.I., H.S. and T.S.; Japan Science and Technology Agency, under the512

ERATO Higashiyama Live-Holonics Project [JPMJER1004] to T.H. and T.S.
22

513
514
515
516

Supplemental materials
The

supplemental

materials

for

this

paper

are

availableat

http://dx.doi.org/10.1080/09168451.2017.1422107.

517
518
519
520

Accession number
FASTQ files of the RNA-seq reads are available with the accession number
DRA006324.

521

23

522

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650

Figure Legends

651

Fig. 1.

Delayed flower opening in JA-insensitive jai1-1 mutants.

652

Note: (A–C) Primary inflorescences of WT Micro-Tom (MT-GH) (A) and jai1-1

653(B, C) plants. (A) and (B) were almost at the same developmental stage because the size654

and shape of the young flower buds were equivalent. (C) was at a later stage around655

three days after (B). Each flower and flower bud was labeled with a lowercase letter (the656largest one was a, followed in order by b, c, d...). A fully opened flower (white squares),657

a partially opened flower (grey squares), a petal-emerged bud (black squares), and a658

smaller bud (no square) are indicated, respectively. (D, E) Developmental progress of659

flower buds in WT (D) and jai1-1 (E). The shapes of all flower buds in a representative660

primary inflorescence were continuously observed once daily at noon. A fully opened

661

flower (white bars), a partially opened flower (grey bars), a petal-emerged bud (black

662bars), and a smaller bud (black lines) were indicated, respectively. (F) Average length of663

the period from petal emergence (when petal exceeds sepal) to fully open, which is the664

sum of the lengths of petal-emerged buds and partially opened flowers. An error bar

665

represents the S.D. n is indicated in each bar. Significance was calculated using an

666

unpaired Student’s t-test. ***P<0.001.

667
668

Fig. 2. Development of WT and jai1-1 flower buds toward opening and changes of669

phytohormone levels in the flower buds. The levels of nine phytohormones in the same670

bud sample were simultaneously measured.

671

Note: (A, B) Morphology of representative flower buds and flowers at indicated672

ages. WT (MT-GH) (A) and jai1-1 (B). Bars = 5 mm. (C) The means of individual

673

hormone levels in three samples of each age and their S.D. are indicated as pmol per

674

gram fresh weight (pmol/gFW). WT (solid line) and jai1-1 (dashed line) were compared.

29

675

Asterisks indicate significant differences between WT and jai1-1 at the same bud age

676(Student’s t-test, P<0.05). n = 3.

677
678

Fig. 3.

Changes of gene expression in WT and jai1-1 flower buds toward opening.

679

Note: (A−C) Comparisons among the changes of gene expressions of SlAOC (A),680

SlOPR3 (B), and SlMYB21 (C) in developing flower buds/flowers. WT (solid lines) and681

jai1-1 (dashed lines) were measured. The highest level of each gene expression in WT

682

was arbitrarily set to 1. DNAJ was used as the internal control. Error bars indicate the683

S.D. Asterisks indicate significant differences between WT and jai1-1 at the same bud684

age (Student’s t-test, P<0.05). n = 3. (D) A neighbor-joining phylogenetic tree showing

685

amino acid sequence similarities among SlMYB21 and closely related R2R3-MYB

686

proteins in tomato and Arabidopsis. AtMYB21, AtMYB24, and AtMYB57 are

687

indicated by asterisks. Bootstrap values are indicated at the roots.

688
689

Fig. 4.

Bud and flower development in AtMYB24-SRDX plants.

690

Note: (A, B) Inflorescences of WT (A) and AtMYB24-SRDX (B) plants. (C−E)

691

Morphology of representative flower buds and flowers at the indicated day number of692

age. WT (C), jai1-1 (D), and AtMYB24-SRDX (E) plants, all of which were in a693

Micro-Tom (MT-GH) background, are compared. Lower panels are an enlargement of694

the inner organs after removal of the front organs. Arrowheads indicate dehisced anthers.

695

Bars = 1 mm.

696
697

Fig. 5.

Abnormal male and female fertility in AtMYB24-SRDX flowers.

698

Note: Abbreviations for the plant samples in this figure are as follows. M8,

699

Momotaro 8; AtMYB24-SRDX, AtMYB24-SRDX transgenic tomato in a Momotaro 8

700background; MT, Micro-Tom (MT-GH); jai1-1, jai1-1 mutant. (A) Cross sections of
30

701

developing fruits. Arrowheads, developing seed; arrows, placenta. Bars = 5 mm. (B)702

Expression levels of the SlMYB21 gene in various floral organs of day-6 Micro-Tom

703

flower buds. Se, sepals; Pe, petals; An, anthers; Fi, filaments; St, styles; Ov, ovaries.

704

SAND was used as the internal control. The level in petals was arbitrarily set to 1. Error

705

bars, SD. n = 3. (C) Pollen tube elongation visualized by aniline blue staining. WT

706

Micro-Tom pistils were manually pollinated with the pollen grains of indicated plant707

samples. Arrows, aborted pollen tube. Bars = 100 µm. (D) DAPI-stained pollen grains

708

corrected from dehisced anthers. Arrowheads, generative cell nucleus; arrows,

709

vegetative cell nucleus; asterisks, tricellular pollen grain. Bars = 100 µm. (E)710

Appearance rate of trinuclear pollen grains. More than 100 pollen grains were counted

711

per assay. Error bars, S.D. n = 3. ** P < 0.01 (Student’s t-test). (F) Pollen tube

712

elongation visualized by aniline blue staining. Pistils of indicated plant samples were713

manually pollinated with WT (M8) pollen grains. Arrows, aborted pollen tube. Bars =714

100 µm. (G) Ovules isolated from opening or equivalent flower buds after treatment715

with clearing solution. Brackets, female gametophyte; arrowheads, micropyle. Bars =716

100 µm.

717

31

(B)	

WT

(C)	

jai1-1a	(F)	

jai1-1

a	

a	
b	

b	

h	

i	

c	

b	

c	
d	

e	
 g	
 f	

Individual flower bud
in the inflorescence

(D)	

i	
h	

g	
f	

c	
d	

Number of days elapsed	
5	
10	
a	
b	
c	
d	
e	
f	
g	
h	
i	

d	

e	
f	

e	

g	

(E)	
15	

WT

a	
b	
c	
d	
e	
f	
g	
h	
i	
j	

h	

Days in petal-emerged bud
and partially opened flower	

(A)	

***	

88	

66	

44	

22	

00	

80	

67	

WT	
 jai1-1	
WT	
  jai1-­‐1	
  Number of days elapsed	
5	
10	

15	

jai1-1

Fig. 1. Delayed flower opening in JA-insensitive jai1-1 mutants.	
Note: (A–C) Primary inflorescences of WT Micro-Tom (MT-GH) (A) and jai1-1 (B, C) plants.
(A) and (B) were almost at the same developmental stage because the size and shape of the young
flower buds were equivalent. (C) was at a later stage around three days after (B). Each flower andflower bud was labeled with a lowercase letter (the largest one was a, followed in order by b, c,d...). A fully opened flower (white squares), a partially opened flower (grey squares), a petalemerged bud (black squares), and a smaller bud (no square) are indicated, respectively. (D, E)
Developmental progress of flower buds in WT (D) and jai1-1 (E). The shapes of all flower buds ina representative primary inflorescence were continuously observed once daily at noon. A fully
opened flower (white bars), a partially opened flower (grey bars), a petal-emerged bud (black bars),
and a smaller bud (black lines) were indicated, respectively. (F) Average length of the period frompetal emergence (when petal exceeds sepal) to fully open, which is the sum of the lengths of petalemerged buds and partially opened flowers. An error bar represents the S.D. n is indicated in eachbar. Significance was calculated using an unpaired Student’s t-test. ***P<0.001. 	

(A)	
WT
1	
 2	
 3	

4	

5	

6	

1	
 2	
 3	

4	

5	

6	

JA

2.E+032

7	

8	

9	

10	

11	

7	
8	
9	
10	
Flower bud age (day number)	

11	

12	

WT

jai1-1(B)	
jai1-1

3.E+043

2.E+042

*	

1
1

22

33

44

66

77

*	

3.E+033

88

ABA

2.E+032

1.E+0310.E+000

1
1

22

33

*	
44

55

66

77

2.E+01288

0.E+000
1
99
1

GA1

2.E+032

*	

*	

*	
1
1

22

33

44

55

66

77

0.E+000
1
99
1

iP

4.E+004x10 pmol/gFW

88

22

33

44

55

66

77

88

0.E+00099
1
1tZ

2.E+052

pmol/gFW

3.E+003

1.E+0112.E+002

*	
22

33

44

55

66

77

88

0.E+000
99
1
1

33

44

55

66

77

88

99

GA4

22

33

44

55

66

77

88

99

SA

1.E+051

1.E+0011122

1.E+031

1.E+0112.E+012

0.E+00099*	*	x105 pmol/gFW

x103 pmol/gFW

55

0.E+000*	

1.E+021x10 pmol/gFW

0.E+000*	

1.E+031*	

IAA

x103 pmol/gFW

1.E+041

JA-Ilex103 pmol/gFW

x104 pmol/gFW*	

2.E+022x102 pmol/gFW

(C)	

22

33

44

55

66

77

88

0.E+000
99
1
1

22

33

44

55

66

77

88

99

Flower bud age (day number)	

Fig. 2. Development of WT and jai1-1 flower buds toward opening and changes of phytohormone
levels in the flower buds. The levels of nine phytohormones in the same bud sample were
simultaneously measured.	
Note: (A, B) Morphology of representative flower buds and flowers at indicated ages. WT (MTGH) (A) and jai1-1 (B). Bars = 5 mm. (C) The means of individual hormone levels in threesamples of each age and their S.D. are indicated as pmol per gram fresh weight (pmol/gFW). WT(solid line) and jai1-1 (dashed line) were compared. Asterisks indicate significant differences
between WT and jai1-1 at the same bud age (Student’s t-test, P<0.05). n = 3. 	

*	Relative level	

(A)	
1.0	
1	
  

WT

SlAOC

*	

*	

0	
  0	
1	
2	
  

2	

AtMYB24	
*	

961	

*	

997	

*	

SlMYB21	
AtMYB57	
*	

3	

3	
  

4	
  

(B)	

4	

5	
  

5	

6	
  

6	

7	
  

7	

8	
  

8	

9	
  

AtMYB78	

1000	

9	

10	
  

AtMYB108	

786	

*	

SlOPR31.0	
1	
  SlMYB78	
*	

1000	

999	
980	

0.5	
0.5	
  

SlMYB108	SlMYB2	

805	

SlMYB112	
735	

0	
  0	
1	
2	
  

2	

3	
4	
  

3	
  

4	
5	
  

5	
6	
  

(C)	
Relative level	

SlMYB28 [Outgroup]	
AtMYB21	
*	

1000	

*	

0.5	
0.5	
  

Relative level	(D)	

jai1-1SlMYB21
1.0	
1	
  

0.5	
0.5	
  

6	
7	
  

7	
8	
  

*	

*	

9	
  8	

AtMYB2	

9	
10	
  

1000	
AtMYB112	
1000	

475	

*	

AtMYB116	AtMYB62	968	

SlMYB62	

*	
344	

0	
  0	
1	
2	
  

SlMYB45	SlMYB116	

473	

SlMYB24	
2	

3	
  

3	
4	
5	
6	
7	
4	
  
5	
  
6	
  
7	
  
8	
  
Flower bud age (day number)	

8	

9	
  

9	

1000	

10	
  

SlMYB57	

0.05	

Fig. 3. Changes of gene expression in WT and jai1-1 flower buds toward opening.	
Note: (A−C) Comparisons among the changes of gene expressions of SlAOC (A), SlOPR3 (B),and SlMYB21 (C) in developing flower buds/flowers. WT (solid lines) and jai1-1 (dashed lines)
were measured. The highest level of each gene expression in WT was arbitrarily set to 1. DNAJ wasused as the internal control. Error bars indicate the S.D. Asterisks indicate significant differences
between WT and jai1-1 at the same bud age (Student’s t-test, P<0.05). n = 3. (D) A neighborjoining phylogenetic tree showing amino acid sequence similarities among SlMYB21 and closely
related R2R3-MYB proteins in tomato and Arabidopsis. AtMYB21, AtMYB24, and AtMYB57 areindicated by asterisks. Bootstrap values are indicated at the roots.	

(A) WT(C) WT
3

4(D) jai1-1
3

4

5

6

7

fully open 
8
9

10

5

6

7

8

9

fully open 
10

6

7

8

9

10

(B)AtMYB24-SRDX(E) AtMYB24-SRDX
3

4

5

13

Fig. 4. Bud and flower development in AtMYB24-SRDX plants.
Note: (A, B) Inflorescences of WT (A) and AtMYB24-SRDX (B) plants. (C!E) Morphology ofrepresentative flower buds and flowers at the indicated day number of age. WT (C), jai1-1 (D), and
AtMYB24-SRDX (E) plants, all of which were in a Micro-Tom (MT-GH) background, arecompared. Lower panels are an enlargement of the inner organs after removal of the front organs.
Arrowheads indicate dehisced anthers. Bars = 1 mm. 

AtMYB24-SRDX (B)Relative expression level

(A)WT (M8)

&#2

WT (MT)

jai1-1SlMYB21

%#1

!#0

An 72#Se
Fi StOv
+3# Pe43# 56#+8# 9:#(C)

WT (M8)(D) WT (M8)

AtMYB24-SRDXWT (MT)*
jai1-1

Tricellular pollen grains (%)

20
&!"#

WT (MT)

jai1-1AtMYB24-SRDX (E)

**

15
%$"#

10
%!"#

$"#5
!"#0

WT AtMYB24 WT'()(*#+,-.# '/#
(M8) -SRDX(MT)

012%# jai1-1(F)

WT (M8)AtMYB24-SRDXWT (MT)

jai1-1WT (M8)AtMYB24-SRDXWT (MT)

jai1-1(G)

Fig. 5. Abnormal male and female fertility in AtMYB24-SRDX flowers.
Note: Abbreviations for the plant samples in this figure are as follows. M8, Momotaro 8; AtMYB24-SRDX, AtMYB24-SRDX transgenic
tomato in a Momotaro 8 background; MT, Micro-Tom (MT-GH); jai1-1, jai1-1 mutant. (A) Cross sections of developing fruits. Arrowheads,
developing seed; arrows, placenta. Bars = 5 mm. (B) Expression levels of the SlMYB21 gene in various floral organs of day-6 Micro-Tom
flower buds. Se, sepals; Pe, petals; An, anthers; Fi, filaments; St, styles; Ov, ovaries. SAND was used as the internal control. The level in petals
was arbitrarily set to 1. Error bars, SD. n = 3. (C) Pollen tube elongation visualized by aniline blue staining. WT Micro-Tom pistils weremanually pollinated with the pollen grains of indicated plant samples. Arrows, aborted pollen tube. Bars = 100 µm. (D) DAPI-stained pollen
grains corrected from dehisced anthers. Arrowheads, generative cell nucleus; arrows, vegetative cell nucleus; asterisks, tricellular pollen grain.
Bars = 100 µm. (E) Appearance rate of trinuclear pollen grains. More than 100 pollen grains were counted per assay. Error bars, S.D. n = 3. **P < 0.01 ;+8<=368>?#!@83?8AB#(F) Pollen tube elongation visualized by aniline blue staining. Pistils of indicated plant samples were manually
pollinated with WT (M8) pollen grains. Arrows, aborted pollen tube. Bars = 100 µm. (G) Ovules isolated from opening or equivalent flower
buds after treatment with clearing solution. Brackets, female gametophyte; arrowheads, micropyle. Bars = 100 µm. 