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Integration of environmental and developmental (or metabolic) control of seed mass by sugar and ethylene metabolisms in Arabidopsis Lai-Sheng Meng, Meng-Ke Xu, Wen Wan, and Jing-Yi Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05992 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 12, 2018

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Journal of Agricultural and Food Chemistry

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Integration of environmental and developmental (or metabolic)

2

control of seed mass by sugar and ethylene metabolisms in

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Arabidopsis

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Lai-Sheng Meng1＊, Meng-Ke Xu1, Wen Wan1, and Jing-Yi Wang1

5

1. The Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, School

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of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, People’s Republic

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of China.

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＊Corresponding author: [email protected].

＊

9 10

Running title: YDA interacts with EIN3 for the seed size control

11 12

ABSTRACT

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In higher plants, seed mass is an important to evolutionary fitness. In this context, seedling

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establishment positively correlates with seed mass under conditions of environmental

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stress. Thus, seed mass constitutes an important agricultural trait. Here, we show

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loss-of-function of YODA (YDA), a MAPKK Kinase, decreased seed mass and lead to

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susceptibility to drought. Furthermore, we demonstrate that yda disrupts sugar

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metabolisms but not the gaseous plant hormone, ethylene. Our data suggest that the

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transcription factor EIN3 (ETHYLENE-INSENSITIVE3), integral to both sugar and

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ethylene metabolisms, physically interacts with YDA. Further, ein3-1 mutants exhibited

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increased seed mass. Genetic analysis indicated that YDA and EIN3 were integral to a

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sugar-mediated metabolism cascade which regulates seed mass, by maternally controlling 1

ACS Paragon Plus Environment


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embryo size. It is well established that ethylene metabolism leads to the suppression of

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drought tolerance by the EIN3 mediated inhibition of CBF1, a transcription factor required

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for the expression genes of abiotic stress. Our findings help guide the synthesis of a model

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predicting how sugar/ethylene metabolisms and environmental stress are integrated at

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EIN3 to control both the establishment of drought tolerance and the production of seed

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mass. Collectively, these insights into the molecular mechanism underpinning the

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regulation of plant seed size, may aid prospective breeding or design strategies to increase

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crop yield.

31 32

Key words: Arabidopsis, Seed Mass, Drought Tolerance, YODA (YDA),

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ETHYLENE-INSENSITIVE3 (EIN3), Ethylene and Sugar Metabolisms.

34 35

INTRODUCTION

36

Seed size/mass is regulated by three important constituents, the seed coat, the endosperm

37

and the embryo1. In angiosperms, a double-fertilization process is involved in seed

38

development, and in this process, a diploid embryo is produced by one sperm nucleus

39

fused with the egg cell; on the other hand, the triploid endosperm is produced via the other

40

fused with two polar nuclei1. With seed maturity of Arabidopsis, the seed forms one layer

41

of endosperm cells, and the seed coat is formed by the maternal integument. The

42

endosperm surrounds the embryo, and the maternal seed coat again surrounds the

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endosperm. Thus, this harmonious growth of zygotic tissues and maternal sporophytic

44

decides seed size.

45

Mutation of the cytokinin receptor results in seeds with twice the mass of wildtype. 2


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Thus, cytokinin may modulate embryo size by a endospermal and/or maternal

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mechanism2,3. The apetala2 (ap2) and auxin response factor2 (arf2) mutants have

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increased seed mass caused by enhancing both embryonic cell size and cell number.

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Furthermore, seed properties are determined by both the maternal sporophyte and

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endosperm genomes4-6. MINISEED3 (MINI3), one member of WRKY (WRKY

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DNA-BINDING PROTEIN) transcription factor, and a leucine rich repeat (LRR) kinase

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IKU2 (HAIKU2) regulate seed mass7. SHB1 (SHORT HYPOCOTYL UNDER BLUE 1)

53

is recruited to promoters of both the MINI3 and IKU2. While the shb1-D overexpression

54

mutant has increased seed mass, a shb1 loss-of-function mutant has reduced seed mass7.

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Seed size is negatively relative to the produced seed number and is positively

56

relative to seedling survival8,9. However, larger seeds generally develop into larger

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seedlings, which are more robust in their tolerance of both abiotic stresses and resource

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deprivation9,10. Therefore, large seed plants are thought be more effective competitors.

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These above observations suggest that a metabolism molecule, either environmental or

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developmental molecule, may involve regulation of both abiotic stresses and seed mass.

61

Therefore, it remains to be established what metabolism molecule integrates plant

62

responses to environmental stresses into the control of seed mass. Currently, a report has

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showed that the AN3-YDA gene cascade is involved in the modulation of drought tolerance,

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and this cascade is independent of ABA11. In this work, we found that Arabidopsis yda

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mutants, which perturb stomatal and embryo development12,13, exhibited small seed mass

66

and comprised drought tolerance in an ABA independent fashion. Our findings suggested

67

that the decrease in yda seed mass was because of a decline in embryo cell size. This was

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found to be a consequence of the suppression of embryo cell elongation; which is typically

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a result of abnormal sugar metabolism. Furthermore, in vitro pull-down assays identified a

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component of both sugar and ethylene metabolisms, ETHYLENE-INSENSITIVE3 (EIN3),

71

as an interactor with YDA. The interaction in the physiological relevance was confirmed

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via co-immunoprecipitation assays with plant extracts. Furthermore, ein3-1 mutants

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exhibited large seed mass due to an embryo of increased size, as a consequence of embryo

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cell elongation. Genetic analysis indicated that YDA-EIN3 formed a sugar-mediated

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metabolism cascade for the regulation of seed mass. It has been reported that EIN3

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modulates the CBFs expressions negatively by binding to specific elements within their

77

promoters, which leads to diminished expression of COR15A14. While the seed mass of

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ctr1 was significantly reduced, that of ein2 and ein3 eil1 mutants was enhanced, which is

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likely a consequence of abnormal embryonic cell elongation. Collectively, these results

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suggest a model where, under water sufficiency, sugar levels enhance cell elongation by

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sugar-specific YDA-EIN3/EIL1 function, while ethylene metabolisms repress cell

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elongation by an ethylene-specific C2H4-Receptors-CTR1-EIN2-EIN3/EIL1 cascade.

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This antagonistic interaction results in the development of large seeds. Conversely, when

84

water deficiency induces a dramatic increase in both sugar and ethylene metabolisms,

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drought resistance is established by the antagonistic interaction between the sugar-specific

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YDA-EIN3/EIL1-CBFs-COR15A metabolism

87

C2H4-Receptors-CTR1-EIN2-EIN3/EIL1-CBFs-COR15A

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producing smaller seeds. Based on above analysis, we explain how environmental and

89

developmental (or metabolic) control of seed mass is integrated by sugar and ethylene

90

metabolisms in Arabidopsis.

cascade

91

4


and

the

ethylene-specific

metabolism

cascade,

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MATERIALS AND METHODS

93 94

Plant materials and growth conditions

95

yda-1, yda-2, and yda-1013,15,16, ein3-1, ein3 eil1, ctr1-1 and ein2-5 mutants17-19,

96

estradiol-inducible EIN3–FLAG19, and estradiol-inducible ∆ N-YDA12 transgenic plants

97

with Col-0 background were described previously.

98

yda-1, yda-2, ein3-1, ein2-5, and eil1-3 were obtained from the ABRC (Ohio State

99

University). Prof H.W Guo (BeiJing University, China) kindly provided the ein3-1 eil1-3

100

and EIN3-FLAG seeds. Prof H.Q Yang (Shanghai JiaoTong University, China) kindly

101

provided the yda-10 seeds and pHB-35Spro-YDA:GFP plasmids.

102

The yda ein3 mutant was obtained from F2 seedlings of yda-10 ein3-1 that mature

103

stomata in the 8-day-old cotyledons are constitutively developed and formed in clusters in

104

the dark16, and had longer hypocotyls grown on solid MS medium supplemented with 3µm

105

ACC for 3 days13. Transgenics plants containing relative plasmid constructs were

106

produced by floral dip method mediated by the Agrobacterium tumefaciens17,35.

107 108

Seed treatment, sowing, growth conditions and environment growth chamber has been described in ref 17.

109 110

Plasmid constructs

111

An EIN3 (At3G20770) promoter-GUS construct were generated via inserting 0.5 kb

112

promoter fragments, which amplified primers (P1-ggg gac aag ttt gta caa aaa agc agg ct

113

AAC AAA TGT GTC GAA GAA CGT G, P2-ggg gac cac ttt gta caa gaa agc tgg gt AGA

114

TCA GGA AGA TAG ATC ATA G) for EIN3. These sections were amplified into

5



115

pCB308R, as has been previously described in ref 11.

116 117

GUS assay

118

GUS assay has been previously described in ref 17.

119 120

Quantitative RT-PCR analysis

121

Total RNA and was Quantitative RT-PCR analysis has been previously described in ref 17.

122

For analyzing ERF1 expression in Col-0, Ler, yda-1 and ctr1-1 developing siliques,

123

primers F-5'-ATG GAT CCA TTT TTA ATT CAG TCC-3' and R-5'- CAT GGC CGT CGT

124

CTT ACG C -3' were used.

125 126

Assay of sugar metabolite

127

Developing seeds at 6, 9 and 12 DAP were used in this experiments. Assay of sugar

128

metabolite has been previously described in ref 17 and 37.

129 130

Cytological experiments

131

Cytological experiments has been previously described in ref 5.

132 133

Enzymatic assay of invertase by ELISA

134

The extraction and purification has been previously described in ref 23. Enzymatic assay

135

of invertase by ELISA has been previously described in ref 17.

136 137

Localization of invertase activities

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Localizing invertase activity has been described in ref 38 with minor modification.

139 140

Protein expression and purification

141

In this experiment, the plasmid pGEX-5X-1 (for EIN3, CINV2, SUC1 and HXK1) and

142

pET28a (for YDA) was used. By using the primer pair (5′- GCGGCCTTTTTGGCC

143

-ATGCCTTGGTGGAGTAAATCAA

144

5′-ATAAGAAT-GCGGCCGC-TTAGGGTCCTCTGTTTGTTGAT-3′), the YDA coding

145

sequence was amplified, and they were then cloned into the Not1 and Sfi1 restriction sites

146

of the pET28a plasmid for forming the plasmid. The CDS in CINV2 was amplified via this

147

pair

148

GGAATTC-TCAGCAAGTCCATGAAGCAGAT-3′) primers, and they were then cloned

149

into the BamH1 and EcoR1 restriction sites of the pGEX-5X-1 plasmid for forming the

150

plasmid.

151

The

-3′

(5′-CGGGATCC-TGGAGGAAGGTCATAAAGAAC-3′

CDS

of

EIN3

was

amplified

via

this

and

and

pair

5

-

(5′-GGATCC

152

ATGATGTTTA ATGAGATGGG -3′ and 5′-CTCGAGTGCTCTGTTTGGGAT-3′) primers,

153

and they were then cloned into the BamH1 and xhoI restriction sites of the pGEX-5X-1

154

plasmid for forming the plasmid. The CDS of HXK1 was amplified via this pair

155

(5′-GGATCCATGGGTAAAGTAGCTGTTGGA-3′

156

5′-CTCGAGTTAAGAGTCTTCAAGGTAGAG -3′) primers, and they were then cloned

157

into the BamH1 and xhoI restriction sites of the pGEX-5X-1 plasmid for forming the

158

plasmid. The

159

ATGGGAGCCTATGAAACAGA-3′ and 5′-CCCGGGCTAGTGGAATCCTCCCATGGT

160

-3′) primers, and they were then cloned into the BamH1 and SmaI restriction sites of the

and

CDS of SUC1 was amplified via this pair (5′- GAATTC

7



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pGEX-5X-1 plasmid for forming this plasmid. Recombinant glutathione S-transferase

162

binding protein (GST)-tagged EIN3, CINV2, HXK1 and SUC1 and recombinant HIS

163

binding protein (HIS)-tagged YDA were drown from transformed E. Coli (Rosetta2) after

164

ten

165

isopropylβ-D-1-thiogalactopyranoside. By using HIS or GST-agarose affinity, respectively,

166

the recombinant proteins were purified.

hours

of

incubation

at

16

°C

after

induction

with

10µM

167 168

In vitro pull-down assay

169

HIS–YDA and GST–EIN3, GST-CINV2, GST-SUC1, GST-HXK1 expression constructs

170

were prepared as described in ref 39. The in vitro interaction between YDA and these GST

171

fusion protein was performed. Briefly, the HIS–YDA fusion and Ni-NTA sefinose resin

172

(Sangon, Shanghai, China) were mixed at 4°C for 2 h of rocking after brief centrifugation

173

to precipitate beads, and washed 3–4 times with PBS buffer supplemented with 0.5%

174

Tween 20. The precipitated beads were mixed with these GST fusion protein or GST in the

175

in vitro binding buffer (1 mM phenylmethylsulfonyl fluorided; 0.15 M NaCl; 50 mM

176

Tris-HCl, pH 7.5; and 1 mM DTT, 0.5% Triton X-100) and incubated at 4°C for 2 h of

177

rocking, followed by brief centrifugation to precipitate beads, and washed 3–4 times with

178

PBS buffer supplemented with 0.5% Tween 20. By using an anti-GST antibody,

179

SDS-PAGE for immunoblot analysis resolved the bound proteins. The reactive bands were

180

visualized via exposure for

181

tetrazolium.

nitrobluing bromochloroindolyl phosphate/nitroblue

182 183

Coimmunoprecipitation analysis

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Coimmunoprecipitation experiments using wildtype and transgenic plant extracts were

185

performed according to ref 39 with minor modification. Transgenic plants harboring both

186

FLAG–EIN3 and GFP–YDA expression constructs were harvested. Dissoluble protein

187

extracts were gained using a protein extract kit (Sangon, Shanghai, China). Protein extracts

188

was mixed with Anti-FLAG M2 Magnetic Beads (Sigma) and incubated at 4°C for

189

overnight of rocking, followed by brief centrifugation to precipitate beads, and washed 3–4

190

times with PBS buffer supplemented with 0.5% Tween 20. The coimmunoprecipitated

191

GFP-YDA were tested by western analysis with anti-GFP (Sigma) antibodies.

192 193

Starch Staining

194

Starch staining in root tip was performed, as has described previously in ref 40.

195 196

RESULTS

197

yda mutants exhibited small seed mass

198

The expression of YDA can be observed in embryo tissues and in yda mutants, zygote

199

elongation is inhibited and embryos develop within a narrow niche of the seed coat13.

200

Collectively, these findings imply that YDA might modulate seed mass. Therefore, we

201

carried out experiments to investigate this possibility. We obtained one mutant: yda-10

202

from ABRC15,16. The seeds of self-pollinated homozygous yda-10 had ~ 30% lower

203

surface area than did wildtype Col-0, whereas the seeds of transgenic seedlings expressing

204

the constitutively active YDA mitogen activated MAPKK kinase ∆N–YDA [∆YDA/+;

205

YDA protein containing no N-terminal fragment (DN–YDA) is constitutively active]13

206

presented ~62% larger area than did wildtype (Col-0) (Figures 1A and C). These findings 9



207

indicate that YDA may regulate seed mass.

208 209

YDA acts on influencing seed mass maternally

210

To gain further insight into the genetic control of seed mass, we determined if YDA acts

211

zygotically or maternally to regulate seed size. Reciprocal crosses between yda-10 and

212

Col-0 plants were performed. When yda-10 or Col-0 pollen were used as donor and yda-10

213

seedlings were employed as the acceptor of pollen, the influence on seed size was not

214

changed with the alternations of pollen donor (Supplemental Table 1). Similarly, when

215

Col-0 plants were utilized as acceptor and yda-10 or Col-0 pollen were utilized as the

216

donor, the influence on seed mass was also not altered along with the alteration of the

217

donor (Supplemental Table 1). Together, these results indicate that yda mutants exert their

218

influence maternally on the development of seed mass.

219

The embryo includes the most volume in a mature Arabidopsis seed and by extension,

220

alternations in seed mass makes be known in the embryo size. Therefore, we mainly

221

focused on the Arabidopsis embryo.

222 223

Small seed mass of yda mutants is due to small embryo cell size and integument area

224

YDA is expressed in embryos and yda mutant zygotes have impaired elongation during

225

embryo development, with the length these mutant embryos approximately half that of

226

wildtype13. Because of the suspensor lack, embryos of yda mutants are grown in a

227

narrow niche of the seed coat above the micropyle and produce a group of irregularly and

228

isodiametric cells with wedge-shape13. These data suggest that the small seeds of yda

229

mutants might be due to a reduction of embryo mass. And then mature embryos were

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isolated and visualized from yda-10, ∆YDA/+ and Col-0 seeds. The mature embryos of

231

yda-10 mutants were smaller relative to those of wildtype (Figure 1B). We further

232

investigated whether the reduction in yda-10 embryo stature was caused by either embryo

233

cell size or number. Cytological experiments indicated that, on average, the cotyledon

234

embryo area in Col-0 was ~1.5 times that in yda-10 (Figures 1B and D); whereas the

235

average area of Col-0 cotyledon embryo cells was ~1.7 times that of yda-10 (Figures 1B

236

and E). Based on these results (i.e. 1.7/1.5 ≥ 1.0), we conclude that the yda-10 embryos

237

were reduced in size due to the suppression of embryo cell elongation. Similarly, we found

238

that the ∆YDA/+ embryos were increased in size because of embryo cell elongation

239

(Figures 1B, D and E). Therefore, YDA might positively control seed size by regulating

240

embryo cell size.

241

In ovules, the size of integuments is well known to effect seed size6. We naturally

242

asked whether YDA functions via the maternal integument to influence the size of seeds.

243

Therefore, the mass of the ovule and outer integument is assayed. We determined mature

244

ovules from both yda-10 and wildtype plants at 2-4 days after emasculation. We also

245

investigated the outer integument size of wildtype and yda-10 seeds after pollination. The

246

size of outer integuments and ovules in wildtype and yda-10 exhibited an obvious

247

difference at 2-4 days after pollination (Supplemental Figure 1). Thus, these findings imply

248

that YDA via the maternal integument for affecting embryo size, and finally leads to effect

249

seed mass.

250 251

yda impacts sugar but not ethylene metabolisms

252

Since YDA is involved in the regulation of embryo size, a question to be asked is the nature

11



253

of the molecular mechanism that mediates this process. yda has abnormal embryo

254

development, stunted seedlings and increased numbers of stomata12,13. This prompted us to

255

investigate the possible link of YDA with general hormone- metabolisms. However,

256

general hormone metabolisms have previously been reported not to restore the growth

257

defects of yda embryo roots13, implying yda function is not associated with hormone

258

metabolisms.

259

Significantly, it has been reported that AN3-YDA forms a gene cascade integral to the

260

regulation of drought tolerance11 and YDA may be involved in sugar metabolism based on

261

gene chip of ref 12, 13. Thus, it is pertinent to determine if yda are sugar metabolism

262

mutants. Delayed flowering suggests this capability of the associated mutation to

263

counteract the affecting sugars on flowering time; indeed, ref 5 found that changed sugar

264

metabolism affected seed mass. We observed that the yda-10 mutant showed early

265

flowering with fewer rosette leaves (Supplemental Table 2). Further, 5 % glucose restored

266

this altered rosette leaf number in yda-10 mutants (Supplemental Table 2). These findings

267

indicate that the abnormal rosette leaf number of yda-10 plants was dependent on sugar

268

metabolisms. In the yda-1 mutant, the phenotype of roots is like that ia as a result of

269

carbon starvation triggered by decreased ability for sucrose catabolism in root cells. These

270

yda phenotypes include extremely decreasing in root growth13 and the starch loss in the

271

root cap (Figures 2D), due to abnormal cell elongation in root elongation zone. We thus

272

suggest that cell elongation in yda-1 roots is deviant due to absence of cell-wall synthesis,

273

but not a deficiency of cell-wall synthesis pathway41. Thus, under abnormal sugar

274

metabolism, the regulation of seed mass in yda mutants might be caused by embryo cell

275

elongation by cell wall modification.

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276

Since cotyledons are derived from embryos, we utilized cotyledons as material for

277

studying the seed embryo cell elongation response to glucose. The yda mutants had smaller

278

cotyledons compared with wildtype on solid MS medium supplemented with 1% sucrose

279

(Figure 2A). However, the abnormal cotyledon size in yda mutants could be restored on

280

MS medium with 5% glucose (Figure 2B). To confirm this, we observed the anatomical

281

structure of the cotyledons. The yda mutants had smaller cotyledon cells relative to Col-0

282

plants on solid MS medium supplemented with 1% sucrose; whereas supplementation with

283

5% glucose led to yda cotyledon cells not significantly different to controls (Figure 2F).

284

Furthermore, protein levels of YDA-GFP was enhanced on MS medium with 5% glucose

285

relative to either 1% sucrose or 5% mannitol (Figure 2C). Similarly, GFP fluorescence in

286

PHB:YDA-GFP hypocotyls was assayed. 5% glucose, but not 5% mannitol, increased the

287

accumulation of YDA-GFP in the nucleus (Figures 2E). These findings suggest that the

288

accumulation of YDA:GFP in the PHB:35Spro-YDA:GFP line in response to glucose is

289

specific and not related to osmotic stress. These results imply, that YDA might regulate

290

sugar-mediated cotyledon cell elongation and thus modulate cotyledon size.

291

An antagonistic interaction between the plant stress hormone ethylene and glucose

292

was uncovered following the phenotypic and genetic analysis of the Arabidopsis

293

glucose-insensitive and glucose-oversensitive mutants18. As our data implies yda might

294

influence sugar metabolisms, we determined if yda also impacts ethylene metabolisms. As

295

expected, ein3-1, a partial ethylene-insensitive mutant18, was insensitive to ethylene,

296

proved

297

1-aminocyclopropane-1-carboxylic acid (ACC) treatment (Figure 3A19). In contrast with

298

the ein3-1 mutant, yda mutants revealed similar shortened hypocotyls to those of wildtype

via

longer

roots

and

hypocotyls

relative

13


to

wildtype

on


299

upon ACC treatment (Figures 3A and B). Agreeing with the phenotypes, the expression

300

levels were comparable between control and yda-1 seedlings of the ethylene response

301

genes, including ERF1, but they were distinct from ctr1 seedlings (Figure 3C). A previous

302

study19 has also demonstrated that the embryo roots of yda-1 and yda-2 mutants were

303

comparable between control and yda-1 seedlings. Thus, our results imply that yda may not

304

be associated with ethylene metabolisms.

305

A previously reported microarray data of yda mutants13 suggested that only 14 of 8000

306

genes had alternation over two-fold in expressions, they included those integral to sugar

307

metabolisms ( AT4G15760, AT2G43570, AT5G57550, and AT3G27660), cell wall

308

synthesis (At2g45220), and sucrose response (AT5G13930). Thus, over half of the

309

differentially regulated genes in yda mutants were closely related to sugar metabolisms. In

310

another microarray study of yda mutants12, 11% of upregulated genes were found to be

311

involved in cell wall differentiation, a process underpinned by sugar utilisation. These

312

results thus further substantiate our data implying YDA might be involved in sugar

313

metabolisms. In addition, the genes that encode glucokinases, including AT1g12080 and

314

AT2g16790, were strikingly differentially regulated in yda plants.

315 316

Taken together, our findings indicate that YDA might function in sugar metabolisms but not ethylene metabolisms.

317 318

The ratio of sucrose to hexose shows a positive association with embryo cell

319

elongation rather than cell division in yda-10 mutants

320

Sucrose cleavage is catalyzed by hydrolysis of mediated by invertase and produces glucose

321

and fructose (hexoses)20. To test the content of these important metabolites, we performed

14


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322

High Performance Liquid Chromatography (HPLC) analysis in yda-10 and Col-0

323

developing seeds. The concentration of glucose and fructose had declined at 6, 9 and 12

324

DAP in yda-10 compared with corresponding Col-0 developing seeds (Figure 4A).

325

However, while the sucrose concentration was not significantly different between 6- and

326

9-DAP developing seeds in yda-10 and Col-0 plants, the concentration was higher in

327

12-DAP yda-10 seeds (Figure 4B). Consequently, this ratio of sucrose to hexose levels was

328

higher in 6- and 12-DAP developing seeds of yda-10 relative to Col-0 plants (Figure 4C).

329

In general, during seed development, a lower ratio of sucrose to hexose is closely related to

330

cell division activity at the early phase; by contrast, a higher ratio of sucrose to hexose is

331

correlated with cell elongation at the late phase5,21,22. Therefore, a higher ratio of sucrose to

332

hexose in yda-10 developing seeds indicates that YDA regulates seed mass through

333

modulating cell elongation but not cell division during the late phase of seed development.

334

This posit is consistent with findings of cytological experiments (Figure 1), which have

335

suggested that the small embryos of yda mutants might be because of the suppression of

336

embryo cell elongation during the late phase of seed development. The above data also

337

imply that YDA has a role in regulating accumulations of soluble sugars during seed

338

development by unknown molecular mechanisms.

339

Since the change in concentration of sucrose and glucose is regarded because of altered

340

invertase activity23, we speculated that invertase activity may be changed in yda-10 mutant

341

seedlings. Neutral invertase is essential to normal growth and development of in seedlings

342

of Arabidopsis41, rice24 and legumes25. To explore if the activity of neutral invertase was

343

changed in yda-10 mutants, neutral invertase protein levels were assayed by Enzyme

344

Linked Immuno Sorbent Assay (ELISA) in developing seeds of these mutants and control 15



345

plants. Neutral invertase activity in yda-10 mutants was reduced by 28% compared to that

346

in Col-0 (Figure 4D). This is consistent with the yda-1 root phenotype which is alike with

347

carbon starvation triggered by decreased ability for sucrose catabolism in root cells (Figure

348

2D13). To confirm this, we determined cell wall invertase activity. We did not observe a

349

nitroblue tetrazolium (NBT) precipitate in the developing seeds of yda-10 mutant plants,

350

but such a precipitate was obviously in the corresponding organ of Col-0 plants (Figure 4E).

351

This data suggests that cell wall invertase had higher activity in Col-0 relative to yda-10 in

352

developing siliques. Taken together, our experiment results indicate that endogenous

353

sucrose accumulation in yda-10 mutants might be the result of reduced neutral invertase

354

activity, which is consistent with the notion that YDA is associated with sugar metabolisms

355

(Figure 2).

356 357

YDA shows interaction with EIN3 in vivo and in vitro

358

It has been reported that key genes involved in sugar metabolisms include EIN3, HXK1,

359

SUC1 and CINV218,41. EMB71/YDA is the Bck1/Ste11/MEKK1 class of MAPKK kinase.

360

Therefore, YDA might regulate EIN3, HXK1, SUC1 or CINV2 at the post-translation level

361

during sugar metabolisms. To identify if any of these proteins are potential target(s) of

362

AN3 and YDA during sugar metabolisms, we performed in vitro pull down experiments.

363

Full-length YDA was expressed as a protein of histidine (HIS) fusion, and full-length EIN3,

364

HXK1, SUC1 and CINV2 were expressed as Gluthione S-transferase (GST) fusion

365

proteins. Following mixing of the fusion proteins under test, sefinose resin was utilised to

366

bind selectively the given HIS fusion protein. The presence of coprecipitated GST fusion

367

proteins was examined using a GST antibody. Our results indicated that YDA bound to

16


Page 16 of 43

Page 17 of 43

368


EIN3 (Figure 5A) but not HXK1, SUC1 and CINV2 (Supplemental Figure 1) in vitro.

369

It is well established that EIN3 is a target of both the sugar and the ethylene

370

metabolism pathways18,26. To confirm YDA binds specifically to EIN3, we performed in

371

vivo pull down experiments. Following transformation and subsequent crossing,

372

transgenic plants expressing both YDA-GFP and EIN3-FLAG were produced. An

373

anti-FLAG antibody was then utilised for immunoprecipitation of EIN3-FLAG.

374

Subsequently, coimmunoprecipitation of YDA-GFP was detected using a GFP antibody

375

(Figure 5B). Therefore, the above data reveal a direct interaction between YDA and EIN3

376

in planta.

377

As YDA is the Bck1/Ste11/MEKK1 class of MAPKK kinase, EIN3 stability/function

378

might be affected by YODA interaction and subsequent phosphorylation. While the two

379

proposed EIN3 phosphorylation sites are thought to show dual functions: T174 for

380

stabilization and T592 for degradation26, the kinase(s) responsible for EIN3

381

phosphorylation remain to be rigorously determined19,26.

382 383

EIN3 acts on influencing seed mass maternally

384

To gain further insight into the genetic regulation of seed size, we investigated if EIN3 acts

385

zygotically or maternally to regulate seed size. Thus, reciprocal crosses between ein3-1

386

and Col-0 plants were performed. When either ein3-1 or Col-0 pollen was utilized as the

387

donor and ein3-1 mutant plants were utilized as an acceptor, the influence on seed size was

388

not changed with the alternation of pollen donor (Supplemental Table 3). Similarly, when

389

ein3-1 or Col-0 pollen were utilized as the donor and Col-0 plants were utilized as an

390

acceptor, seed size was also not changed by the alternations of donor (Supplemental Table

17



391

3). Together, these results indicate that EIN3 exhibits an effect on seed size maternally.

392 393

ein3-dependent Increased Seed Mass is mediated by Enhanced Embryo Cell Size

394

EIN3, a key component of ethylene metabolisms, negatively regulates stem elongation and

395

leaf expansion in mature plants19, implying that EIN3 might regulate cell proliferation.

396

Therefore, we assayed if seed size in ein3 mutants is altered relative to that of wildtype.

397

Our findings presented that the ein3-1 had larger mature seeds than wildtype (Figures 6A

398

and C). Further, on average, cytological experiments revealed the embryo area of ein3-1

399

cotyledons was ~ 1.20 times that of wildtype (Figures 6B and D); while the area of ein3-1

400

cotyledon cells was ~1.40 times that of wildtype (Figures 6B and E). In terms of these

401

results (i.e. 1.20/1.40 ≤ 1), we concluded that ein3-1 embryos were enlarged due to

402

increased embryo cell size. Thus, EIN3 controls embryo size by regulating embryo cell

403

elongation, similar to YDA. In addition, we determined EIN3 expression in reproductive

404

organs by employing a β-glucoronidasae reporter gene driven by the EIN3 promoter

405

(ProEIN3:GUS). Our results indicated that GUS activity was detected in flowers, pistils,

406

stamens, testa, embryos and siliques (Supplemental Figure 2). Hence, the profile of EIN3

407

expression agrees with a potential function for this gene in the regulation of seed mass.

408 409

EIN3 acts downstream of YDA genetically in controlling seed size

410

Double-mutant analysis was performed, which is combining ein3-1 (large seeds) with

411

yda-10 (small seeds), for assaying if YDA acts upstream of EIN3 in controlling seed mass.

412

We selected yda-10 ein3-1 plants for further analysis. The yda-10 ein3-1 mutant had

413

similar seed mass to ein3-1 (Figures 6A and F), indicating ein3-1 was epistatic to yda-10. 18


Page 18 of 43

Page 19 of 43


414

Therefore, our results indicate that YDA functions genetically upstream of EIN3 in

415

modulating seed mass.

416 417

Analysis of ctr1, ein2 and ein3eil2 mutants reveals that changes in seed mass

418

correlates with embryonic cell size

419

On average, the epidermal cell area of leaf blades in ctr1 mutants was reduced to ~1/5 of

420

wildtype (Col-0), consequently, the leaf area was dramatically reduced in the ctr1

421

mutant27,28. In addition, the ein3eil1 and ein2 mutants showed large leaf blades and

422

expanded stems19. Therefore, we investigated whether the seed mass in these mutants is

423

altered compared with that of wildtype. Our results revealed that seed mass of ctr1 plants

424

was significantly reduced, whereas seed mass of the ein3eil1 and ein2 mutants was

425

increased compared with that of wildtype (Figures 7A and D). Further analysis found that

426

reduction of ctr1 seed mass was due to reduction of embryonic cell size, because of the

427

inhibition of embryonic cell prolongation. In contrast, the increase of ein3eil1 and ein2

428

seed mass was a consequence of increased embryonic cell size, due to embryonic cell

429

elongation (Figures 7B, C, E and F). Since CTR1-EIN2-EIN3/EIL1 comprises the

430

canonical ethylene metabolisms and this hormone regulates cell proliferation by cell

431

elongation but not cell division29,30, ethylene metabolisms may regulate cell elongation in

432

these mutants.

433

434

DISCUSSION

435

In this work, we use three kinds of lack of YDA, that is, yda-10 (SALK_105078C), yda-2

436

(CS6393), and yda-1(CS6392)13. Both alleles result in abnormal phenotypes during 19



437

embryonic period, and homozygous yda-1 and yda-2 cannot produce normal seeds13.

438

However, homozygous yda-10 can produce normal seeds16. In details, yda-10, yda-1 and

439

yda-2 have been described in ref 13, 16.

440 441

YDA and EIN3 regulate seed mass by sugar-mediated embryo cell elongation by

442

modulating sugar metabolisms

443

The ein3 and yda mature embryos were obviously larger and smaller relative to those of

444

wildtype, respectively (Figures 1 and 6). It is well established that embryo mass is resolved

445

by both embryo cell size and cell number. In terms of cytological data, our results implied

446

that decreased yda embryo mass was because of suppression of embryo cell elongation and

447

increased ein3 embryo size was a consequence of embryo cell elongation. Further analysis

448

revealed that the alternation of cell elongation in the yda and ein3 embryos is triggered by

449

sugar metabolisms; which is based on comparison of glucose response phenotypes in

450

cotyledons, the stability of YDA in high glucose concentrations, the ability of glucose to

451

reverse delayed flowering in the yda mutant and the loss of starch from the root cap in yda

452

plants.

453

In general, during seed development, a lower ratio of sucrose to hexose is closely

454

related to cell division activity at the early phase. In contrast, a higher ratio of sucrose to

455

hexose is correlated with cell elongation at the late phase5,21,22. It appears that the ratio of

456

sucrose to hexose is more important than the absolute concentrations of sucrose and

457

hexose5. When embryos were of immature fava bean cultured in hexose with high

458

concentrations, they undertook cell division. Conversely, embryos cultured in high

459

concentrations of sucrose performed cell elongation21. Thus, a lower ratio of sucrose to

20


Page 20 of 43

Page 21 of 43


460

hexose generates a result for cell division, whereas a higher ratio produces a result for cell

461

elongation. In the early stage of seed development (6 DAP) (Figure 4), there was a lower

462

ratio of sucrose to hexose, which generated a result for cell division. In the middle and later

463

stages of seed development (9 and 12 DAP) (Figure 4), there was a higher ratio of sucrose

464

to hexose, which generated a result for cell elongation. Further analysis indicated that

465

accumulation of aberrant endogenous sucrose was caused by abnormal neutral invertase

466

activity in yda mutants (Figure 4). Together, the above results suggest YDA regulates seed

467

mass by sugar-mediated embryo cell elongation by modulating sugar metabolisms.

468 469

YDA-EIN3 may regulate seed mass via protein–protein interactions

470

While seed mass of yda mutants showed smaller mass than that of control wildtype, seed

471

mass of ein3-1 mutants presented larger size than that of control wildtype (Figures 1 and

472

6). Furthermore, seed mass of yda-1ein3-1 double mutant had larger mass than that of

473

control wildtype (Figure 6), implying EIN3 is downstream of YDA and loss of EIN3

474

function significantly enhanced seed mass in yda mutants. These ﬁndings suggest a

475

negative relationship between YDA and EIN3 during regulating seed development.

476

Further, data showing a YDA interaction with EIN3 in vitro and in vivo (Figures 5A and

477

B) and sugar sensing (Figures 2A and B18), the neutral invertase activity (Figures 4D and

478

E; Supplemental Figure 3), protein stability in high concentration of glucose (Figures 2C

479

and E18), provided additional support for possible negative regulatory activities of YDA

480

upon EIN3. Collectively, these data suggest that a YDA interaction with the EIN3 might

481

regulate seed mass.

482

Currently, the precise mechanism via which YDA regulates EIN3 is unknown,

21



483

however, we have found that YDA directly interacts with EIN3, which is a common

484

target of both ethylene and sugar metabolisms or/and sugar signaling18. As YDA is the

485

Bck1/Ste11/MEKK1 class of MAPKK kinases, EIN3 stability/function might be affected

486

following YDA interaction and subsequent phosphorylation. The two proposed EIN3

487

phosphorylation sites are thought to show dual functions: T174 for stabilization and T592

488

for degradation26, but the kinase(s) responsible for EIN3 phosphorylation remain to be

489

rigorously determined19,26.

490 491

YDA-EIN3 may function as a molecular junction point that integrates modulation of

492

seed growth and development into drought stress responses of plants

493

Mutant plants of an3 exhibit drought tolerance and lower anthocyanin accumulation under

494

drought conditions, phenotypes which are independent of abscisic acid (ABA), whereas

495

yda mutant plants show more sensitivity to drought stress (Supplemental Figure 411).

496

Moreover, on placement to 10% PEG 6000 (polyethylene glycol), a common stress

497

treatment for mimicking drought tolerance in the lab, an3 mutant show larger cotyledon

498

size (seed mass)31, whereas yda mutant presents smaller cotyledon size (seed mass)

499

(Supplemental Figure 4; Figure 1), suggesting drought tolerant with larger seeds.

500

Therefore, the gene cascade of AN3-YDA is a key component for the modulation of both

501

seed mass and drought tolerance11. As has been shown above, seedlings with large seeds

502

are usually more healthy and strong and present a better tolerance to drought stresses,

503

relative to smaller-seeded seedlings8,9. At the macroscopic level, ecologists observed the

504

survival advantage of small-seeded and large-seeded species, and they elucidated the

505

tradeoff tactics between competition and colonization in which species with

22


Page 22 of 43

Page 23 of 43


506

smaller-seeded property are prior to colonizers and species with larger-seeded property are

507

prior to competitors8,9. However, their molecular mechanisms are unknown. Our results

508

suggest this may be explained (Figure 8).

509 510

A proposed model illustrating how integrating environmental and developmental (or

511

metabolic) control of seed mass by sugar and ethylene metabolisms in Arabidopsis

512

The antagonistic interreaction between the plant stress hormone ethylene and glucose is

513

revealed by the phenotypic and genetic analysis of Arabidopsis glucose-oversensitive and

514

glucose-insensitive phenotypes18. Here, besides sugar metabolisms regulating seed mass,

515

we observed ethylene metabolisms function in seed mass regulation. The components

516

CTR1-EIN2-EIN3/EIL1 constitute the canonical ethylene metabolisms (Figure 8). This

517

metabolism system, is required for ethylene mediated-suppression of cell elongation in leaf

518

blades, stems and hypocotyls27,28,33. The ein3-1, ein2-1, and ein3eil1, as ethylene

519

insensitive mutants, exhibited increased seed size as a consequence of embryonic cell

520

elongation. On the contrary, the ctr1-1, as constitutive ethylene response mutant, exhibited

521

small seed size because of the suppression of embryonic cell elongation. Thus, ethylene

522

participates in seed mass regulation via the C2H4-Receptors-CTR1-EIN2-EIN3/EIL1 gene

523

cascade.

524

It is well established CTR1 (CONSTITUTIVE TRIPLE RESPONSE)-EIN2-EIN3/EIL1

525

forms an ethylene metabolism cascade and that EIN3 negatively regulates CBFs which

526

positively modulate expression of the cold responsive marker gene, COR15A14. Ref 14

527

analyzed the main genes based on the function class from public microarray experiments

528

in

the

double

mutant

ein3

eil1

23


(http://www.ebi.ac.uk/


Page 24 of 43

529

arrayexpress/experiments/E-GEOD-18631) and found a subset of 98 genes that

530

participated in the plant responses to environmental stress. For example, CBF1, CBF3,

531

COR15a, and COR15b. The findings were confirmed via Q-RT-PCR14. Further, it was

532

shown that, EIN3 modulates environmental stress negatively via directly modulating the

533

CBF expression14. CBF1 is a transcriptional activator, and it encodes an AP2 domain that

534

can bind to the C-repeaty DRE in response to deficit of water 33. CBF1 can bind to the

535

COR15A promoter34. During cold, acclimation can induce COR15A33. Therefore, AN3

536

probably regulates

537

AN3-YDA-EIN3/EIL1-CBFs-COR15A gene cascade (Figure 8).

plant

responses

to

drought

stress

by the

sugar-specific

538

Our model suggests that under water deficiency both ethylene (ET) and sugar

539

signalling are activated, with the latter requiring AN3 and YDA function. These two

540

signal systems converge at EIN3, an important integration point for developmental (e.g.

541

sugar signalling) and environmental signals (e.g. water deficiency). Our model suggests

542

sugar signalling is dominant over ET signalling, which is consistent with data reporting

543

the depletion of EIN3 under stress conditions16,21. Subsequently, this is predicted to result

544

in the induction of the transcription factor CBF1-3, which drives the stress expression

545

effector genes leading to the tolerance of drought stress. Further, as EIN3 is depleted, the

546

repression on seed mass development is released, enabling the production of larger and

547

more stress tolerant seeds.

548

This model is further supported by data herein showing that an3 mutants, which

549

exhibit drought tolerance12, develop larger cotyledons31 and by extension possess larger

550

seeds15. Further, an3 seedlings showed enhanced seedling establishment under

551

drought-like conditions, as exemplified by increased cotyledon size17. In addition, yda

24


Page 25 of 43


552

mutants which are more sensitive to drought (Supplemental Figure 4), produce smaller

553

seeds (Figure 1A, B and C).

554

In aggregate, our models predicts sugar signals produced from PET (photosynthetic

555

electron transport), which induces the MBW (MYB/bHLH/ TTG1) complex, tune seed

556

mass

557

EMB71/YDA-EIN3-EIL1-CBFs-COR15A signaling pathway. Further, the impact of ET

558

on

559

C2H4-Receptors-CTR1-EIN2-EIN3/EIL1-CBFs-COR15A

560

interferes with the sucrose specific EMB71-EIN3/EIL1-CBFs-COR15A pathway at the

561

EIN3 node.

and

seed

drought

mass

tolerance

and

drought

by

tolerance

the

is

sucrose

mediated signal

specific

by

pathway,

the which

562

In higher plants, seed size is key traits to evolutionary fitness. Seed mass/size

563

reveals a very key trait in agriculture. Large seed mass of agricultural crops not only

564

presents yield improvement but also imply the increase of other merits, for example,

565

sugar beet seed42, blueberry seed oils43, seed biotinylated protein44, seed protein and oil5,

566

seed defense45.

567 568

ASSOCIATED CONTENT

569

Supporting Information

570

Supplementary Figure 1

571

In vitro, YDA, but not HXK1, SUC1 and CINV1, directly interacts with EIN3.

572

HIS-YDA immobilized on amylose resin pulls down (PD) GST-EIN3, and then and by immunoblotting

573

(IB) using an anti-GST antibody, this complex is analyzed.

574


575

EIN3 expression in the organs of reproductive growth. proEIN3:GUS can express on flowers (A), 25



576

stamens(B), pistils (C), embryos (D) and siliques (E).

577


578

(A). Drought resistance of the yda and wildtype (Ler) seedlings, which is after identical water

579

deprivation, and then is identical re-watering periods. Seedlings were taken photo at the time points of

580

ten days with the deprivation of water at three days after re-watering.

581

(B) Survival rate in yda and wildtype (Ler) plants. (A) after re-watering, plant survival rates were

582

assayed. The values are the mean + SD of two independent experiments (***P < 0.001).

583

Supplemental Figure 4.

584

(A). Representative cotyledons of 8-day-old yda-10, an3-4 and Col-0 plants grown on MS medium

585

supplemented with 1.0 % sucrose. In this experiment, using cotyledons as materials, they are on

586

exposure to 10% PEG (polyethylene glycol 6000)40.Magnifications are the same.

587

(B). The difference in the cotyledon size was showed by bar graph in (A) (**P < 0.01, n=12). Col-0 is

588

set as 100%.

589

Supplemental Table 1

590

Crosses of Reciprocal between yda-10 mutants and wildtype plants were analyzed. Plants were

591

manually pollinated. Means ± SD are shown (**P < 0.01). At least 5 seedlings were assayed for each

592

data point.

593


594

Numbers are average leaf growth ± SD (*P < 0.05). for each datum point, 15 to 25 seedlings were

595

measured. After emergence of first flower, rosette leaf was counted.

596


597

Crosses of Reciprocal between ein3-1 mutants and wildtype plants were analyzed. Plants were

598

manually pollinated. Means ± SD are shown (**P < 0.01). At least 5 seedlings were assayed for each

599

data point.

600 601

AUTHOR INFORMATION 26


Page 26 of 43

Page 27 of 43


602

Corresponding Authors

603

*(L.-S.M.) E-mail: [email protected].

604 605

Author Contributions

606

L-SM designed experiments. L-SM, W.W, and J-Y W performed the experiments. L-SM, W.W and

607

M-KX completed statistical analysis of data. L-SM wrote, edited and revised this manuscript.

608 609

Notes

610

The authors declare no competing or financial interests.

611 612

Finding

613

This study was supported by grants from the Agricultural High Technology Research of Xuzhou City

614

(KC16NG063). The Doctoral Scientific Research Founding of Jiangsu Normal University.

615 616

ACKNOWLEGMENTS

617

We thank Prof Gary J Loake ( The University of Edinburgh, Edinburgh, UK) for editing English

618

language.

619 620

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Figure Legend Figure 1. YDA Regulates Seed Mass. (A). Representative mature dry seeds of Col-0 (a), ∆YDA/+(b), and yda-10 (c), respectively. Bar = 1.0 mm for (a) to (c). [18] generated a construct expressing ∆N–YDA fused to a chemical-inducible promoter, XVE.YDA protein lacking the N-terminal fragment (∆N–YDA) is constitutively active, and the growth and development 31



of these transgenic seedlings expressing ∆N–YDA are severely suppressed, and its homozygotic plants cannot flower14. (B). (a), (b) and (c): Representative embryos derived from Col-0 (a) ∆YDA/+(b), and yda-10 (c), respectively. (d), (e) and (f): Representative the panes in Col-0 (a), ∆YDA/+(b), and yda-10 (c) are amplified, respectively. Bars = 100 µm for (a), (b) to (c). Bars = 10 µm for (d), (e) to (f). (C). Bar graph exhibiting the difference in average dry seed area among Col-0, ∆YDA/+, and yda-10 seeds (**P < 0.01, *P < 0.05; n=3, every group has 100 seeds). Col-0 is set as 1.0. (D). Bar graph exhibiting the difference in the embryo area among Col-0, ∆YDA/+, and yda-10 seeds. (**P < 0.01, *P < 0.05; n=20). Col-0 is set as 1.0. (E). Bar graph exhibiting the difference in the embryo cell area among Col-0, ∆YDA/+, and yda-10 seeds. (**P < 0.01; n=50). Col-0 is set as 1.0. Data in (C), (D) and (E) are means ± SD from at least 10 independently propagated Col-0 and mutant lines. Figure 2. The Phenotype Defects of yda Mutant Cotyledons Can Be Restored by High Glucose Concentration. (A). Bar graph exhibiting the difference in the cotyledon area between Ler, yda-1, and yda-2 seedlings grown on solid MS medium with 1% sucrose (**P < 0.01, n=15). (B). Bar graph exhibiting the difference in the cotyledon area between Ler, yda-1,and yda-2 seedlings grown on solid MS medium with 5% glucose (n=15). (C). Representative 5% glucose treatment stabilizes EMB71/YDA protein. The 12-day-old PHB:35Spro-YDA:GFP seedlings grown on solid MS medium with 1% sucrose, 5% mannitol and 5% glucose. Materials were from at least 10 independently propagated lines. (D). Longitudinal sections of roots of 8-day-old Ler (a) and yda-1 (b) seedlings. Seedlings were from the same plate. Magnifications are the same. Results are typical of those for many seedlings. The arrow indicates starch grains. Bar = 50 µm. (E). Representative 1% sucrose, 5% mannitol, and 5% glucose treatment promotes YDA-GFP protein accumulation in the nucleus. The hypocotyls of 12-day-old PHB:YDA-GFP seedlings grown on solid MS medium with 1% sucrose, 5% mannitol, and 5% glucose. White arrows point to GFP-positive nuclei. Magnifications are the same. (F). Bar graph exhibiting the difference in the cotyledon cell size between Ler and yda-1 seedlings grown on solid MS medium with 1% sucrose and 5% glucose, respectively. (*P < 0.05, n=40). Figure 3. YDA is not Involved in Ethylene Signaling. (A). Representative 4-day-old ein3-1, yda-1, yda-2 and Ler seedlings grown on solid MS medium without ACC; and 4-day-old ein3-1, yda-1, yda-2 and Ler seedlings grown on solid MS medium with 3.0 umACC; and 4-day-old ein3-1, yda-1, yda-2 and Ler seedlings grown on solid MS medium with 6.0 umACC; Bar = 5.0 mm.

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(B). Bar graph exhibiting the difference in the hypocotyl length between Ler, yda-1, yda-2 and ein3-1 seedlings grown on solid MS medium with 0.0 um ACC, 3.0 umACC and 6.0 umACC, respectively. Error bars represent SD (n = 16). Experiments were repeated three times with similar results. (C). Bar graph exhibiting the difference of expression of ERF1 between 8-day-old Ler, yda-1 and ctr1-1 light-grown seedlings treated with or without ethylene (25 ppm) for 5 h. Data were from quantitative RT-PCR.Error bars indicate SD (n=3).And wildtype without ethyleneis set as 1.0.Quantifications were normalized to the expression of UBQ5. Figure 4. Sugar Metabolite Analysis. (A). Bar graph exhibiting the difference in the concentrations of hexose (glucose and fructose) between Col-0 and yda-10 seeds (*P < 0.05, n=3). (B). Bar graph exhibiting the difference in the sucrose concentrations between Col-0 and yda-10 seeds (*P < 0.05, n=3). (C). Bar graph exhibiting the difference in the ratio of the sucrose/hexose between Col-0 and yda-10 seeds. (D). Bar graph exhibiting the difference in the neutral invertase activity between developing seeds of Col-0 and yda-10 plants (**P < 0.01, n=3). In (A), (B), (C) and (D), indicated days are days after pollination. (E). Representative nitroblue tetrazolium (NBT) precipitation in the developing seeds of Col-0 (a) and yda-10 (b)lines. Magnifications are the same. Data for (A) to (E) are means± SD from at least 10 independently propagated Col-0 and yda-10 lines. Seeds were removed from siliques before sugar analysis and NBT analysis. Figure 5. YDA Interacts with EIN3 for Regulating Seed Mass. (A). HIS-YDA fusing protein exhibited specific affinity for GST-EIN3 but not GST in vitro. (B). GFP-YDA showed specific affinity with FLAG-EIN3 in vivo. FLAG-EIN3 was associated with membranes and can be detected with anti-GFP antibodies. Wildtype was used as a negative control. Figure 6. EIN3 Regulated Seed Mass through Sugar-Mediated Cell Elongation. (A). Representative mature seeds of WT (a), yda-10 (b), ein3-1 (c) and yda-10ein3-1 (d), respectively. Bar = 0.5mm for (a) to (d). (B). (a) and (b): Representative cotyledon embryos derived from Col-0 (a) and ein3-1 (b) mature seeds. (c) and (d): Representative the panes in (a) and (b) are amplified, respectively. Bars = 10um.

Bars = 100um for (a)

to (b). Bars = 10 um for (c) to (d). (C). Bar graph exhibiting the difference in seed area between WT and ein3-1 seeds (**P < 0.01, n=20). (D). Bar graph exhibiting the difference in cotyledon embryo areas between WT and ein3-1 seeds. (**P < 0.01, n=20). (E). Bar graph exhibiting the difference in the cell area of cotyledon embryos between WT and ein3-1 seeds. (**P < 0.01, 33



n=30). (F). Bar graph exhibiting the difference in average seed area between WT (Col-0), yda-10, ein3-1 and yda-10ein3-1 seeds (**P < 0.01; *P < 0.05, n=30). Data in (C), (D), (E) and (F) are means+ SD from at least 10 independently propagated WT and mutant lines. Figure 7. the ein2 and ein3eil1 Present Large Seed Mass and the ctr1 Present Smaller Seed Mass than Did Wildtype. (A). Representative mature dry seeds of ctr1-1(a), ein2-1 (b), ein3-1eil1-3 (c) and Col-0 (b), respectively. Bar = 0.5 mm for (a) to (d). (B). Representative cotyledon embryos from ein2-1 (a), ein3-1eil1-3 (b), Col-0 (c) and ctr1-1(d), respectively. Bar = 100 um for (a) to (d). (C). Representative cotyledon embryo ein2-1 (a), ein3-1eil1-3 (b), Col-0 (c) and ctr1-1(d), respectively; which is from (B). Bar = 10 um for (a) to (d). (D). Bar graph exhibiting the difference in average seed weight/100 seeds between ein2-1, ein3-1eil1-3, Col-0 and ctr1-1 seeds (**P < 0.01, n=3). (E). Bar graph exhibiting the difference in cotyledon embryo areas between ein2-1, ein3-1eil1-3, Col-0 and ctr1-1 seeds (**P < 0.01; ***P < 0.001, n=20). (F). Bar graph exhibiting the difference in cell areas of cotyledon embryos between ein2-1, ein3-1eil1-3, Col-0 and ctr1-1 seeds (**P < 0.01, n=40). Data in (D), (E) and (F) are means +SD from at least 10 independently propagated WT and mutant lines. Figure 8. A Proposed Model Illustrating. The data described herein, together with our previous data and that reported by others, guides the synthesis of a model linking changes in water status with drought tolerance and seed size.

Seedling establishment under environmental

stress, including drought, positively correlates with seed size. However, in the absence of a stress environment, small seed size associated with increases seed number, conveys a competitive advantage5,9,10.

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a TOC Graphic: A Proposed Model Illustrating how seedling establishment positively correlates with seed mass under conditions of environmental stress.

The data described herein, together with our previous data and that reported by others, guides the synthesis of a model linking changes in water status with drought tolerance and seed size. Seedling establishment under environmental stress, including drought, positively correlates with seed size. However, in the absence of a stress environment, small seed size associated with increases seed number, conveys a competitive advantage5,9,10.


Integration of Environmental and Developmental (or Metabolic

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