Key Metabolic Pathways Involved in Seed Primary Physiological Dormancy

bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Short title: metabolic activities during dormancy maintenance

2 Key metabolic pathways involved in seed primary physiological dormancy

3 maintenance of Korean pine seed1

4 Yuan Song2, Xiaoye Gao

5 College of Eco-Environmental Engineering, Guizhou Minzu University, Guiyang, China

6 550003

7 One-sentence summary: A larger metabolic switch in dormant seeds after 4 weeks of incubation

8 under favorable conditions for germination may maintain primary physiological dormancy of

9 Korean pine seeds.

10 Author contributions: Y.S. wrote the article and prepared the figures. X.G. co-wrote and edited

11 the article. Y.S. agrees to serve as the author responsible for contact and ensures communication.

1 This work was supported by the National Natural Science Foundation of China (31901300,

31960636) and Natural Science Foundation of Guizhou Province (2019) No. 1165.

2 Author for contact: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

19 ABSTRACT

20 The metabolic changes that occurred during either cold stratification or after-ripen treatment, and

21 in both dormant seeds and after-ripened seeds either under the dry state or during imbibition have

22 been extensively explored. Much less is known about those present in both dormant seeds and

23 cold stratified seeds during the same period of incubation under favorable germination conditions.

24 Metabolite composition was investigated in both embryo and megagametophyte of primary

25 physiological dormant seeds (PPDS) of Pinus Koreansis collected at 0 week, 1 week, 2 weeks, 4

26 weeks and 6 weeks of incubation, and of cold stratified seeds with released primary physiological

27 dormancy (RPPDS) sampled at 0 week and 1 week of incubation, seed coat rupture stage and

28 radicle protrusion stage. Embryo contained higher levels of most metabolites compared to

29 megagametophyte. Strong metabolic changes occurred at 1 week and 4 weeks of incubation in

30 PPDS, with most metabolites were significantly accumulated in 4-weeks-incubated PPDS. A

31 larger metabolic switch was found in RPPDS between 1-week-incubation and seed coat rupture

32 stage. Especially, there was a significant major decrease in the relative levels of most

33 phosphorylated sugars and amino acids. The carbohydrate metabolism, especially pentose

34 phosphate pathway and tricarboxylic acid cycle were more active pathways in the embryos of

35 4-weeks-incubated PPDS, but the operation rate of most amino acid metabolism was lower

36 compared to 1-week-incubated RPPDS. We suggest that a larger metabolic switch in the embryo

37 of PPDS after 4 weeks of incubation may assist in maintaining primary dormancy.

2 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

41 Woody plant species determines the structure of forest ecosystem and thus has a profound

42 impact on the ecological conditions found within forest (Zaynab et al., 2018). Seed germination is

43 a key stage in the plant life cycle. Non-dormant seeds germinate under favorable conditions for

44 seedling establishment, beginning the second round of plant life cycle (He et al., 2011). Those

45 orthodox seeds are dry after maturity (De Gara et al., 2000). Seed germination process can be

46 divided three phases based on the characteristic of triphasic mode of water uptake (Bewley, 1997,

47 Finch-Savage and Leubner-Metzger, 2006). A rapid and passive water uptake by dry seeds first

48 occurs during phase I. Then, there is a slow water uptake in phase II during which metabolic

49 activities resume, preparing for subsequent seed germination (He et al., 2011b). The occurrence of

50 further water uptake during phase III is accompanied by the radicle protrusion through seed coat.

51 Seed germination sensu stricto refers to the physiological process spanning from the uptake of

52 water by dry seed to radicle protrusion (Bewley, 1997). For some species, seeds are non-dormant

53 after dispersal from mother plant and rapidly germinate when encounter favorable conditions.

54 However, to survive in unfavorable climates, plants have evolved a seed dormancy mechanism

55 that allow seeds to persist for prolonged periods until conditions for seedling establishment

56 become favorable (De et al., 2019). Seed dormancy can be released either by cold stratification (a

57 low-temperature storage of imbibed seeds) or by after-ripening (a period of dry storage) (Bewley

58 et al., 2012) depending on species and environmental conditions experienced by seed during

59 maturation.

60 Seed germination is a very complex physiological and biochemical process that is mainly

61 determined by genetic factors with a substantial environmental influence (Joosen et al., 2013).

62 Previous studies have revealed that a series of physiological and biochemical process such as

3 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

63 repair mechanisms, transcription, translation, reactivation of metabolism, reserves mobilization,

64 redox homeostasis regulation and organellar reassembly is initiated in imbibed seeds (Bewley,

65 2013; He et al., 2011b). Germination is driven by these metabolic processes. The combination of

66 high-throughput and large scale-omics methods is used to unveil the regulation mechanism of seed

67 germination. Due to the great potential in agriculture and the availability of genome sequence data,

68 the genomic, transcriptomic, and proteomic studies of seed germination have largely focused on

69 model species, such as Arabidopsis (Nakabayashi et al., 2005; Holdsworth et al., 2008a, b; Narsai

70 et al., 2017a), rice (Narsai et al., 2017b), maize (Sekhon et al., 2013), barley (Lin et al., 2014), and

71 wheat (Yu et al., 2014). These studies revealed that one fourth of stored mRNA are

72 metabolism-related in dry seeds, and metabolism-related genes are also highly expressed during

73 germination (Nakabayashi et al., 2005). Metabolism-related proteins are the major groups detected

74 in germinated seeds (He et al., 2011b; Sano et al., 2012). The central carbon metabolism pathways

75 provide energy and building blocks for various metabolic activities (Rosental et al., 2014). It has

76 been suggested that glycolysis, fermentation, the tricarboxylic acid (TCA) cycle and the oxidative

77 pentose phosphate pathway (PPP) are activated during seed germination, with energy is mainly

78 provided by anaerobic respiration, such as fermentation at the early stage of germination (He et al.,

79 2011a, b; Yang et al., 2007). Despite recent great advances in omics studies of seed dormancy and

80 germination, few experiments focus on metabolome-level changes during seed dormancy

81 maintenance and germination in woody plants.

82 Several studies have been conducted to explore the changes in the proteome or metabolome

83 during either cold stratification or after-ripen for the determination of metabolic activities involved

84 in seed germination mechanisms (Lee et al., 2006; Angelovici et al., 2011; Arc et al., 2012;

85 Pawłowski and Staszak, 2016; Chang et al., 2018). It is the metabolic activities occurred before

86 radical protrusion that are responsible for the delay or no germination of seeds (Bewley et al.,

87 2013). In addition, it has been previously shown that water imbibition itself can trigger some

88 various in gene expression or the entire metabolism whatever the depth of seed dormancy (Preston

89 et al., 2009; Yazdanpanah et al., 2017; Xia et al., 2018b). Thus, in order to determine the real

90 physiological and biochemical process involved in seed germination regulation, it is necessary to

91 make dormant and non-dormant seeds to imbibe water for same time during a time window in

92 which radicle has not yet to break through the seed coat. There are also comparative proteomic,

93 transcriptomic, or metabolomic studies generally that are performed on dormant seeds and

94 after-ripened (AR) seeds either under the dry state or during imbibition (Cadman et al., 2006;

95 Chibani et al., 2006; Gao et al., 2012; Joosen et al., 2013; Lu et al., 2016; Yazdanpanah et al., 2017;

96 Xia et al., 2018b; Xu et al., 2020). The mechanisms controlling seed germination are well

97 understood by comparing imbibed dormant and AR seeds. However, seed dormancy release of

98 many species depends on cold stratification. While there has been no study that investigates the

99 metabolic changes in both dormant seeds and cold stratified seeds following transfer to

100 germinative conditions. Fait et al. (2006) have shown that the relative levels of TCA-cycle

101 intermediates, 2-oxoglutarate and isocitrate, 3-phosphoglycerate, fructose 6 phosphate, and

102 glucose 6 phosphate, increased during vernalization and were further enhanced during germination

103 sensu stricto, but how the metabolites in dormant seeds change over imbibition time are still

104 unknown.

105 It has been reported that different tissues of the seed such as embryo and endosperm, have

106 specific functions during seed germination (Baskin and Baskin, 2004; Lee et al., 2006; Xu et al.,

107 2016). Many of previous studies do not investigate the metabolic changes occurring in different

108 seed tissues due to a limitation of sample separation techniques (Ferrari et al., 2009). The real

109 differential metabolic activities between dormant and non-dormant seeds are possibly ignored

110 given the use of mixed tissues over the period of experiment. This requires in future work

111 distinguishing properly the roles of embryo from the endosperm in either dormancy release and

112 subsequent germination or dormancy maintenance.

113 Korean pine is a dominant species in Mixed-broadleaved Korean pine forests (MKPF) in

114 northeast China. Korean pine seeds are physiological dormancy caused by an endogenous block in

115 the embryo. In our previous metabolome study of dormant and cold stratified Korean pine seeds,

116 most metabolites in embryo substantially increased in both dormant and cold stratified seeds

117 during the first 5 days of incubation under favorable conditions. After 5 days of incubation, cold

118 stratified seeds exhibited a lager decrease in the relative levels of almost all amino acids and the

119 TCA-cycle and glycolysis intermediates, while most of metabolites in dormant seeds were

120 maintained at a relative steady level. In the study mentioned above, whole experiment was only

121 carried out for 11 days making it impossible to determine if the embryo of dormant seeds continue

122 to maintain a relatively slow and stable metabolic state with increasing incubation duration.

123 Furthermore, it is still unclear whether those metabolites with decreased relative levels in the

124 embryos of cold stratified seeds show a continuous decrease or a surprising increase during the

125 period from 11 days of incubation to testa rupture and further radicle protrusion. Based on

126 previous those results, it has been previously hypothesized that oxygen consumption would

127 largely decrease through reducing TCA and glycolysis in the embryos of cold stratified seeds rates

128 when compared to dormant seeds. Thus, more oxygen might be supplied to the megagametophytes

129 and used to produce sugars, preparing for rapid germination. However, in the studies

130 mentioned-above, the metabolic changes that occur in megagametophyte were not determined.

131 Thus, the objective of this study was to compare metabolomic profiles of embryo and

132 megagametophyte from dormant dry seeds and seeds with released primary dormancy during

133 incubation under favorable conditions, and reveal key metabolic processes involved in seed

134 dormancy maintenance and germination.

135 RESULTS

136 PCA of the relative contents of metabolites in PPDS

137 PCA was performed on PPDS or RPPDS to investigate metabolic processes at global level.

138 The first component of PCA clearly separated embryo samples (PPDS-0-E, PPDS-1-E, PPDS-2-E,

139 PPDS-4-E and PPDS-6-E) and megagametophyte samples (PPDS-0-M, PPDS-1-M, PPDS-2-M,

140 PPDS-4-M and PPDS-6-M), accounting for 41.6% of the variance. PPDS-0-E and PPDS-0-M

141 samples formed two more discrete groups on the first principal component (Fig. 1A). The

142 transition from 1 WAI (week after incubation) to 2 WAI stage was associated with smaller

143 differences in the relative of contents of metabolites in both embryos and megagametophytes. The

144 samples collected at 4 WAI and 6 WAI were also situated relatively closely to one another. The

145 metabolome of both embryo and megagametophyte in PPDS started to deviate at 4 WAI on the

146 second principal component.

147 PCA of relative contents of metabolites in RPPDS

148 RPPDS embryo experienced relatively larger metabolome changes than megagametophyte

149 during incubation (Fig. 1B). Embryo did not display distinct alterations in metabolome after 1

150 week of incubation. However, there was a large metabolic shift after seed coat rupture, indicated

151 by the apparent separation between RPPDS-1-E and RPPDS-SCR-E samples along the first

152 principal component. A relatively small difference was found between RPPDS-1-M and

153 RPPDS-SCR-E samples.

154 PCA of the relative contents of metabolites in the embryo and megagametophyte of both

155 PPDS and RPPDS

156 PPDS-4-E sample was clearly differentiated from RPPDS-1-E, RPPDS-SCR-E and

157 RPPDS-RP-E samples on the first dimension of the PCA (Fig. 1C). A similar distinction was also

158 found between RPPDS-1-M, RPPDS-SCR-M, RPPDS-RP-M and PPDS-4-M samples (Fig. 1D).

159 Fold changes of important metabolites with a VIP value > 1 and P < 0.05 during incubation

160 of PPDS

161 When comparing the patterns of change in the relative contents of metabolites in embryos

162 with VIP >1 and P < 0.05 between successive time points, a large variation occurred in the first

163 week (43 increased, 44 decreased) (Fig. 2A), with few changes observed between 1 and 2 WAI (4

164 increased, 16 decreased) (Fig. 2B) and between 4 and 6 WAI (20 increased, 14 decreased) (Fig.

165 2D). There was a relatively higher alternation between 2 and 4 WAI (45 increased, 14 decreased)

166 (Fig. 2C). Those metabolites that displayed a major increase in their relative contents after 1 week

167 of incubation were mainly involved in major carbohydrate metabolism. 6-phospho-D-gluconate,

168 D-mannose 1-phosphate, 3-phospho-D-glycerate, Beta-D-fructose-6-phosphate,

169 phosphoenolpyruvate, ribulose 5-phosphate, glycerol 3-phosphate, D-fructose 1,6-bisphosphate,

170 fructose 1-phosphate increased 5- to 23-fold between 0 and 1 WAI (Fig. 2A). Of PPP

171 intermediates, D-ribose 5- phosphate also increased after 2 WAI (Fig. 2B). In contrast, several

172 sugar alcohols, including ribitol, D-sorbitol, myo-inositol and maltitol exhibited a significant

173 decline in their relative levels at the 1 WAI time point (Fig. 2A). The relative amounts of uridine

174 5'-diphosphate, cytidine 5'-monophosphate, uridine diphosphate glucose,

175 s-methyl-5'-thioadenosine and uridine 5'-monophosphate were significant lower in

176 2-weeks-incubated PPDS as compared to 4-weeks-incubated PPDS (Fig. 2C). The abscisic acid

177 relative content did not change significantly during the first 2 weeks of incubation, and strongly

178 increased 2-fold after 4 weeks (Fig. 2C). Two TCA-cycle intermediates (citrate and cis-aconitate)

179 and three phosphorylated sugars including D-fructose 1,6-bisphosphate, D-ribulose 5-phosphate

180 and alpha-D-galactose 1-phosphate were also significantly accumulated from 2 WAI to 4 WAI

181 (Fig. 2C). Moreover, the relative level of citrate, both the number and relative contents of

182 phosphorylated sugars also increased in 4-weeks-incubated PPDS when compared to

183 1-weeks-incubated PPDS (Fig. 2E). Only 15 metabolites with VIP > 1 and P < 0.05 exhibited

184 large differences between 1 WAI and 6 WAI (Fig. 2F).

185 The relative amounts of most metabolites were significant higher in embryos than that in

186 megagametophytes at all six time points (Fig. 3). A rather similar pattern of metabolites was found

187 in megagametophyte between successive time points, with a large metabolic change between 0

188 and 1 WAI (Fig. 4A) and between 2 and 4 WAI (Fig. 4C). A lesser extent of change in metabolites

189 occurred between 1 and 2 WAI (Fig. 4B) and between 4 and 6 WAI (Fig. 4D). After 1 week of

190 incubation, Beta-D-fructose 6-phosphate and ribitol increased 14-fold and decreased 13-fold,

191 respectively (Fig. 4A). In addition, 13 amino acids (L-aspartate, L-glutamine L-threonine, etc.)

192 rapidly increased 1- to 6-fold (Fig. 4A). The changes in phosphorylated sugars and amino acids

193 were relatively little between 2 and 4 WAI (Fig. 4C). However, the relative levels of most fatty

194 acids were declined 1 to 6-fold from 2 WAI to 4 WAI (Fig. 4C). Higher numbers of amino acids

195 and TCA-cycle intermediates were found to be significantly accumulated between 1 and 6 WAI

196 than between 1 and 4 WAI (Fig. 4, E and F).

197 Fold changes of important metabolites with a VIP value > 1 and P < 0.05 during incubation

198 of RPPDS

199 The relative contents of metabolites in the embryos of RPPDS changed little over the first 1

200 week of incubation (16 increased, 23 decreased) (Fig. 5A). The relative levels of 67 metabolites

201 were significantly reduced in RPPDS with seed coat rupture compared with 1-week-incubated

202 RPPDS (Fig. 5B). In detail, ten phosphorylated sugars (D-mannose 1-phosphate,

203 6-phospho-D-gluconate, Beta-D-fructose 6-phosphate, alpha-D-glucose 1-phosphate, D-ribose

204 5-phosphate, D-ribulose 5-phosphate, D-mannose-6-phosphate, L-fucose-1-phosphate, D-fructose

205 1,6-bisphosphate and fructose 1-phosphate), two sugar alcohols (D-sorbitol and myo-inositol), two

206 disaccharide (D-maltose and trehalose), two trisaccharide (raffinose and stachyose) and 12 amino

207 acids displayed 1- to 8-fold decrease (Fig. 5B). It was found that a surprising 33-fold increase in

208 the relative levels of taxifolin (Fig. 5B). A number of significant changes in metabolite abundance

209 were observed between seed coat rupture and radicle protrusion stages (76 of the 188 metabolites

210 displayed significant changes) (Fig. 5C). There was a 58- and 23 -fold decrease in the respective

211 relative levels of glutathione disulfide and glutathione (Fig. 5C). Beta-D-fructose 6-phosphate,

212 D-mannose 1-phosphate and 6-phospho-D-gluconate dramatically increased 3- to 4- fold (Fig. 5C).

213 However, the differences between the two stages were more subtle compared with the large

214 changes in the metabolome observed from 1 WAI to seed coat rupture stage (Fig. 5, B and C).

215 At 0 WAI, 1 WAI and seed coat rupture stage, the embryos in RPPDS contained higher

216 relative contents of most metabolites than megagametophytes (Fig. 6). At 1 WAI, a smaller

217 proportion of the detected metabolites with VIP > 1 and P < 0.05 changed in megagametophytes

218 (Fig. 7A). The trend of changes in metabolites was then followed by the largest change between 1

219 WAI and seed coat rupture stage (Fig. 7B), followed by a relatively small change between seed

220 coat rupture and radicle protrusion stages (Fig. 7C). During the period form 1 WAI to seed coat

221 rupture stage, two sugar alcohols (maltitol and myo-inositol), one disaccharide (D-maltose), and

222 three trisaccharide (raffinose, stachyose and maltotriose) significantly decreased 1- to 5-fold (Fig.

223 7B). While most of the changes in amino acids were seen to occur in megagametophyte, 15 amino

224 acids displaying 1.4- and 2.7-fold increases at seed coat rupture stage (Fig. 7B).

225 Fold changes of important metabolites with a VIP value > 1 and P < 0.05 between PPDS-4W

226 and RPPDS-1W, PPDS-4W and RPPDS-SCR

227 The metabolic process occurred before radicle protrusion regulate the complete of

228 germination. In addition, the large metabolic changes in PPDS were observed after 4 weeks of

229 incubation. Thus, we performed the fold change analyses of metabolites differentially accumulated

230 between 1-week-incubated RPPDS, RPPDS with seed coat rupture and 4-weeks-incubated PPDS,

231 aiming at reveal real potential metabolic processes that control seed primary physiological

232 dormancy maintenance. 92 metabolites were found to be significantly accumulated in the embryos

233 between PPDS-4W and RPPDS-1W (Fig. 8A). Of these, the relative levels of 41 metabolites were

234 significant higher in PPDS-4W, including 6-phospho-D-gluconate, abscisic acid, raffinose,

235 stachyose, alpha-D-glucose 1,6-bisphosphate, Beta-D-fructose 6-phosphate, D-ribulose

236 5-phosphate, D-mannose 1-phosphate, D-fructose 1,6-bisphosphate and citrate, etc. The relative

237 levels of most amino acids were higher in RPPDS-1W than that in PPDS-4W. Only the relative

238 levels of 27 metabolites were significantly different in embryos between RPPDS-SCR and

239 PPDS-4W (Fig. 8B). 6-phospho-D-gluconate, D-mannose 1-phosphate and Beta-D-fructose

240 6-phosphate accumulated 10- to 48-fold in PPDS-4W. However, taxifolin and 3-dehydroshikimic

241 acid decreased 62- and 86-fold, respectively, in PPDS-4W compared to RPPDS-SCR.

242 The relative content of abscisic acid in megagametophyte was 5.3-fold higher in PPDS-4W

243 rather than in RPPDS-1W (Fig. 8C). D-maltose, raffinose, D-lyxose, myo-inositol, D-fructose,

244 ribitol and sucrose also remained high relative levels in PPDS-4W, whereas 14 amino acids were

245 at least twofold more highly accumulated in RPPDS-1W compared with PPDS-4W. More amino

246 acids were remained in the megagametophytes of RPPDS-SCR than in PPDS-4W (Fig. 8D). In

247 contrast, raffinose, stachyose, D-maltose, maltotriose, ribitol, myo-inositol, D-fructose, sucrose,

248 maltitol and sedoheptulose exhibited higher relative contents in PPDS-4W.

249 The patterns of change in metabolic pathways during incubation of PPDS and RPPDS

250 Most metabolites whose relative levels significantly changed during incubation of PPDS or

251 RPPDS were mainly associated with carbohydrate metabolism, amino acid metabolism and lipid

252 metabolism (Table 1 and 2). The activities of the above-mentioned most metabolic processes were

253 higher in embryos than that in megagametophytes at the various time points of incubation. For

254 PPDS, a dramatic change in most metabolic pathways was observed at both 1 WAI and 4 WAI

255 (Table 1). There was also a strong metabolic switch from 1 WAI to seed coat rupture stage in

256 RPPDS (Table 2).

257 Differential metabolic pathways between PPDS-4 and RPPDS-1, between PPDS-4 and

258 RPPDS-SCR.

259 Carbohydrate metabolism and amino acid metabolism were the mainly changed metabolic

260 pathways between PPDS-4 and RPPDS-1, between PPDS-4 and RPPDS-SCR (Table 3). There

261 were more differential metabolic pathways in embryo between PPDS-4 and RPPDS-1 than

262 between PPDS-4 and RPPDS-SCR.

263 DISCUSSION

264 This study provides a comprehensive profile of the metabolites during germination of Korean

265 pine seeds. After dry PPDS were imbibed for 1 week under favorable conditions, most metabolites

266 in embryos with significant FC and VIP > 1 were involved in carbohydrate metabolism and amino

267 acid metabolism. In detail, there was a great increase in the relative levels of

268 6-phospho-D-gluconate, D-mannose 1-phosphate, 3-phospho-D-glycerate, Beta-D-fructose

269 6-phosphate, phosphoenolpyruvate, ribulose 5-phosphate, D-fructose 1,6-bisphosphate and

270 fructose 1-phosphate associated with pentose phosphate pathway, fructose and mannose

271 metabolism and glycolysis. This in accordance with our previous studies that showed the

272 glycolytic pathway activated in the dormant Korean pine seeds upon imbibition (Song and Zhu,

273 2019). It has been reported that a rapid uptake of water occurred during first 5 days of incubation

274 of dry dormant Korean pine seeds. The increase in various metabolic activities during this period

275 might be caused by water uptake. Further investigations about the metabolic changes that occur in

276 dormant seeds with the extension of incubation time are thus needed to distinguish the

277 consequences of water absorption from the metabolic activities involved in seed dormancy

278 maintenance. We found that these early responses to water were then followed by relatively small

279 changes in the relative contents of metabolites between 1 and 2 WAI. In contrast, there was a

280 larger metabolic switch occurs as the incubation time was extended to 4 weeks, with the relative

281 levels of 45 of the 59 metabolites with significant FC and VIP > 1 were increased between weeks

282 2 and 4 of incubation. Furthermore, both PPP and TCA ran at a higher rate at the 4 WAI time point

283 when compared to 1 WAI. In addition, glycolysis-cycle-related metabolites, D-fructose

284 1,6-bisphosphate and alpha-D-glucose 1-phosphate, were also accumulated. However, this

285 accumulation did not last until the 6 WAI time point. A transient but rapid increase in most

286 metabolites occurred after 4 weeks of incubation when dormant seeds had already been imbibed.

287 Based on these findings, we proposed that dormant Korean pine seeds are far from metabolically

288 quiescent state when they were in favorable conditions for germination of non-dormant seeds.

289 Cadman et al. (2006) also found that transcriptional activities took place in primary dormant (PD)

290 Arabidopsis Cvi seeds after 30 days of imbibition due to 1833 genes were highly expressed

291 compared with 24-h-imbibed PD seeds.

292 Only a small proportion of the metabolites such as L-alanine, L-aspartate and N-(L-arginino)

293 succinate, were differentially accumulated in the embryos of RPPDS during the first 1 week of

294 incubation. However, the relative levels of those metabolites involved in the carbohydrate

295 metabolic pathways including fructose and mannose metabolism, TCA cycle, PPP, galactose

296 metabolism and starch and sucrose metabolism were significantly reduced. The pattern of change

297 in metabolic activities described above is contrary to our earlier statements that Korean pine seeds

298 rapidly resumed metabolic activity upon incubation in suitable conditions for germination (Song

299 and Zhu, 2019). In the present study, when RPPDS were retrieved from virgin MKPF forest in late

300 May, these seeds had previously experienced the relatively high temperature conditions in soil,

301 which might be the reason for the slow increase in the relative levels of the most metabolites

302 under laboratory conditions. That is, a great metabolites increase was not observed in the present

303 study because of the delay in sampling time. In the embryos of RPPDS with seed coat rupture,

304 there was a major decrease in the relative levels of various phosphorylated sugars (such as

305 D-mannose 1-phosphate, 6-phospho-D-gluconate, Beta-D-fructose 6-phosphate, alpha-D-glucose

306 1-phosphate, D-ribulose 5-phosphate, etc.), polysaccharides (D-maltose and trehalose),

307 oligosaccharides (raffinose and stachyose) and most amino acids when compared to

308 1-week-incubated RPPDS. Based on these results we hypothesize that the metabolic activity is

309 attenuated in the embryos of RPPDS with seed coat rupture. Moreover, TCA-cycle was also

310 significantly down-regulated relative to RPPDS. Non-Targeted metabolomics analysis of Poplar

311 seeds also revealed that three TCA cycle intermediates (citrate, isocitrate and succinate)

312 substantially accumulated during the periods of slow water uptake and then promptly decreased

313 until radicle protrusion (Qu et al., 2019). Research conducted with non-dormant and dormant

314 seeds of Leymus chinensis demonstrated that the proteins involved in TCA cycle were decreased

315 in non-dormant seeds (Hou et al., 2009). When the radicle protruded through the seed coat, part of

316 the embryo was in an atmosphere containing sufficient oxygen, PPP was significantly enhanced,

317 while TCA cycle did not exhibit dramatic change. Allen et al. (2010) suggested that fermentation

318 still proceeded in Arabidopsis seeds approaching radicle protrusion.

319 It is generally accepted that glycolysis, fermentation, the TCA cycle and PPP are activated to

320 provide energy for metabolic activity during seed germination of non-dormant seeds (Fait et al.,

321 2006; Preston et al., 2009; Angelovici et al., 2011; Weitbrecht et al., 2011; He and Yang, 2013;

322 Zaynab et al., 2018). It has been also suggested that because of limited oxygen or lack of

323 functional mitochondria, energy is mainly provided by glycolysis and fermentation during early

324 stage of germination (Yang et al., 2007; He et al., 2011b; He and Yang, 2013; Ren et al., 2018;

325 Zaynab et al., 2018), and TCA cycle is the main energy source at the late stage of germination. A

326 decrease in TCA cycle activity would lead to the inhibition of germination of dormant seeds

327 (Chibani et al., 2006; Angelovici et al., 2011; Das et al., 2017; Ren et al., 2018). However, we

328 found that the relative levels of a number of metabolites related to carbohydrate metabolism were

329 significant higher in the embryos of 4-weeks-incubated PPDS, especially those related to PPP and

330 TCA cycle, when compared to 1-week-incubated RPPDS. Xia et al. (2018a) also detected a higher

331 activity of enzymes related to TCA cycle and glycolysis in imbibed-dormant sunflower seeds.

332 Furthermore, sunflower seed dormancy can be released by cyanide (a respiratory inhibitor),

333 implying the importance of respiratory activity in seed dormancy maintenance (Oracz et al., 2008;

334 2009).

335 Conversion of glucose through glycolysis, β-oxidation and TCA cycle yields nicotinamide

336 adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2) which serve as

337 electron donors for the generation of adenosine triphosphate (ATP) in the mitochondrial electron

338 transport chain (Liemburg-Apers et al., 2015). The electron escaped from electron transport chain

339 (ETC) can lead to the generation of reactive oxygen species (ROS) (Liemburg-Apers et al., 2015).

340 NADPH-oxidases are also involved in the production of ROS (Ye et al., 2012). Although it has

341 been documented that ROS accumulation in the cells might damage proteins and membranes,

342 ROS also act as a key positive regulatory factor in the activation of germination process (Murthy

343 et al. 2003; Bailly, 2004; Oracz et al., 2007; El-Maarouf-Bouteau and Bailly, 2008; Müller et al.,

344 2009; Parkhey et al. 2012; Ye et al., 2012). Sugar enters to glycolysis and further TCA cycle

345 mainly through phosphorylation of sugars (Fait et al., 2006). A significant increase of numerous

346 phosphorylated sugars and organic acids involved in glycolysis, PPP and TCA cycle during

347 incubation of PPDS may indicate an overaccumulation of ROS, leading to the maintenance of

348 seed dormancy. There was a 1- and 10-fold increase in the respective levels of adenosine

349 triphosphate (ADP) and adenosine monophosphate (AMP) in 4-weeks-incubated PPDS compared

350 with 1-week-incubated RPPDS. In addition, the ratio of glutathione to glutathione disulfide was

351 also slightly higher in 4-weeks-incubated PPDS. These results suggested that the PPDS might be

352 subjected to more severe oxidative stress condition. In contrast, the activities of TCA cycle and

353 PPP are attenuated most presumably to reduce the generation of ROS during incubation of RPPDS

354 under favorable conditions for germination. The reduced TCA cycle and PPP may reflect a way to

355 avoid energy waste and reserve consumption, preparing for the establishment of autotrophic

356 growth of seedling after seed germination.

357 It has been confirmed that abscisic acid (ABA) plays a critical role in the induction and

358 expression of seed dormancy and the inhibition of seed germination (Ali-Rachedi et al., 2004; Liu

359 et al., 2013). Xia et al. (2018b) reported that the ABA level significantly accumulated in dormant

360 sunflower seeds, but continued to decrease in non-dormant seeds after 24 h of imbibition. The 24

361 h imbibition was also the time when the metabolism of non-dormant seeds was clearly

362 differentiated from that of dormant seeds. A rather similar pattern of change in the relative levels

363 of ABA was found in the present study, with a great increase in embryos of PPDS after 4 weeks of

364 incubation and a rapid decline in embryos of RPPDS after 1 week of incubation. Similarly, there

365 was a great variation in metabolism between the 4-weeks-incubated PPDS and 1-week-RPPDS. It

366 is therefore to hypothesize that ABA may affect seed dormancy (or seed germination) process by

367 its action on metabolic actives. Metabolic, transcriptomic and proteomic analyses on seeds treated

368 with exogenous ABA also showed that the regulation of ABA in seed germination is involved in

369 the inhibition of some metabolic processes (Chibani et al. 2006; Xia et al., 2018b).

370 In the present study, all measured metabolic pathways were more active in embryo than that

371 in megagametophyte in both PPDS and RPPDS. Carbohydrate metabolism changed similarly to

372 amino acid metabolism in embryo during incubation. However, in megagametophyte, amino acid

373 metabolism was altered much more prominently than carbohydrate metabolism. In addition, there

374 was a more dramatic change in amino acid metabolism in megagametophytes between 4-weeks

375 incubated PPDS and 1-week-incubated RPPDS. It appears that both carbohydrate metabolism and

376 amino acid metabolism are more predominant metabolic pathways in embryo, but in

377 megagametophyte amino acid metabolism become dominant. Similarly, several studies have

378 demonstrated that the embryos mainly contain enzymes that related to ATP synthesis, energy

379 generation or citrate synthesis (Penfield et al., 2006; Galland et al., 2017; Liu et al., 2018;

380 Domergue et al., 2019). In contrast, catabolic enzymes were found in the endosperm. Xu et al.

381 (2016) reported that proteins with increased abundance in embryo were mainly associated with

382 glycolysis and TCA cycle, cell growth and division and protein synthesis during dormancy release

383 of wild rice seeds. While, the abundances of these proteins in endosperms maintained steady-state

384 level or decreased.

385 The relative levels of amino acids in either embryos or megagametophytes were always

386 significant higher in RPPDS than that in PPDS during incubation. A great decrease in most amino

387 acids occurred from 1 week after incubation to seed coat rupture in the embryos of RPPDS,

388 suggesting that amino acids might be utilized for protein biosynthesis required for seed

389 germination. In contrast, only a few amino acids exhibited a slow increasing trend in embryos

390 during incubation of PPDS. Thus, we concluded that the attenuated amino acid metabolism may

391 maintain primary physiological dormancy in Korean pine seeds. Previous studies have shown that

392 protein biosynthesis was inhibited in dormant seeds compared with non-dormant seeds (Bove et

393 al., 2005; Cadman et al. 2006; Carrera et al., 2008).

394 MATERIALS AND METHODS

395 Seed collection

396 In late September 2018, fresh Korean pine seeds were collected from virgin MKPF in

397 Wuying fenglin National Nature Reserves in northeastern China (128°58′–129°15′E, 48°02′–

398 48°12′N). Seeds contained a low amount of water (about 10%). These fresh dry seeds were then

399 stored at -20 oC to maintain their primary physiological dormancy status.

400 Seed burial experiment

401 In 20 October 2018, a proportion of Korean pine seeds were buried between litterfall and soil

402 in virgin MKPF to release primary physiological dormancy under the effect of lower late autumn

403 and winter temperatures in natural conditions. At the same time, another proportion of seeds were

404 continually kept at -20oC. These seeds were considered as primary physiological dormancy

405 (PPDS).

406 Incubation experiment

407 In late May 2019, seeds were retrieved from virgin MKPF and considered as seeds with

408 released primary physiological dormancy (RPPDS). Both PDS and RPPDS were used for

409 incubation experiment. Seeds were incubated in a growth incubator under a temperature

410 fluctuation regime of 25oC for 14h and 15oC for 10h. An approximately 200 μmol of photons m-2

411 s-1 was provided by cool white fluorescent tubes during high temperature (14h) and darkness was

412 set during low temperature (10h). Group of 20 PPDS (or RPPDS) were placed in a 10-cm Petri

413 dish with 5 layers of filter paper moistened with 10 ml deionized water. Petri dish was then

414 covered with parafilm to reduce water loss. PPDS were incubated for six weeks and sampled on

415 week 0, week 1, week 2, week 4 and week 6 after incubation. The PPDS collected at five time

416 points were abbreviated to PPDS-0, PPDS-1, PPDS-2, PPDS-4 and PPDS-6, respectively. RPPDS

417 were incubated and sampled at four stages (0 week of incubation, 1 week of incubation, seed coat

418 rupture stage and radicle protrusion stage). The RPPDS collected at four stages were abbreviated

419 to RPPDS-0W, RPPDS-1W, RPPDS-SCR and RPPDS-RP, respectively. At each sample time point,

420 five Petri dishes were taken out from growth incubator and used as five replicates.

421 Seed samples pretreatment

422 The relative levels of metabolites in the embryos of five PPDS samples (PPDS-0-E,

423 PPDS-1-E, PPDS-2-E, PPDS-4-E and PPDS-6-E), the megagametophytes of five PPDS samples

424 (PPDS-0-M, PPDS-1-M, PPDS-2-M, PPDS-4-M and PPDS-6-M), the embryos of four RPPDS

425 samples (RPPDS-0-E, RPPDS-1-E, RPPDS-SCR-E and RPPDS-RP-E) and the

426 megagametophytes of four RPPDS samples (RPPDS-0-M, RPPDS-1-M, RPPDS-SCR-M and

427 RPPDS-RP-M) were all used for a non-targeted metabolome analysis. The excised embryos (or

428 megagametophytes) were immediately frozen in liquid nitrogen and subsequently stored at -80oC.

429 Embryos (or megagametophytes) samples were ground into powder under the protection of liquid

430 nitrogen. 80 mg embryo (or megagametophyte) powders were placed inside an Eppendorf tube.

431 One ml of precooled extraction solution (methanol/acetonitrile/ddH2O (2:2:1, v/v/v)) was then

432 added. The mixture of powder and extraction solution was then subjected for following treatments,

433 vortex for 60 seconds, low-temperature ultrasonic for 30 minutes (twice), stand at -20oC for 1 hour

434 to precipitate protein, centrifugation for 20 minutes (14,000 RCF, 4°C). After that, the supernatant

435 was lyophilized and stored at - 80oC until used for further metabolomics analysis.

436 LC-MS analysis

437 The UHPLC/MC system consisted of an Agilent 1290 Infinity LC (Agilent Technologies,

438 Santa Clara, CA, USA) connected to a TripleTOF 6600 mass spectrometer (AB SCIEX, USA).

439 Chromatographic analyses were performed using a hydrop interaction liquid chromatography

440 (HILIC) column (ACQUITY UPLC BEH Amide 2.1 mm × 100 mm column, internal diameter 1.7

441 μm, Waters, Ireland) maintained at 25°C, and the autosampler tray temperature was maintained at

442 4 °C. Mobile phase A was water with 25 mM ammonium acetate and 25 mM ammonia; mobile

443 phase B was acetonitrile. The gradient conditions were 0-1 min, 95% B; 1-14 min, from 95 to 65%

444 B; 14-16 min, from 65 to 40% B; 16-18 min, 40% B; 18-18.1 min, from 40 to 95% B and 18.1-23

445 min, 95% B. The flow rate was 0.3 ml minute-1, and the injection volume was 2μL. For both

446 modes of operation, the mass spectrometric parameters were like that used in Zhou et al. (2019).

447 Data analysis

448 Raw data (wiff. scan files) were converted to mzXML format using ProteoWizard. Then,

449 peak alignment, retention time correction, and peak area extraction was conducted with XCMS

450 program.

451 Metabolite structure identification was performed with accurate mass number matching (< 25ppm)

452 and secondary spectrum matching. The ion peaks with greater missing values (>50%) were

453 removed. Metabolites were searched in a laboratory self-built database. Statistical analyses were

454 conducted with Metaboanalyst (https://www.metaboanalyst.ca/).

455 Principal component analysis (PCA) was used to globally investigate the metabolic changes

456 in both embryo and megagametophyte during incubation of PPDS (or RPPDS). For PCA analysis

457 performed on the relative contents of metabolites in the embryos and megagametophytes of five

458 types of PPDS, the metabolite data were log transformed (generalized logarithm transformation)

459 and auto scaled (mean-centered and divided by the standard deviation of each variable) for

460 normalization. To gain a better normalization result, pareto scaling (mean-centered and divided by

461 the square root of the standard deviation of each variable) was only used for the metabolite data

462 obtained from the embryos and megagametophytes of four types of RPPDS. We also used PCA to

463 analyze the alterations in metabolic profiles of the embryos (or megagametophytes) of both PPDS

464 and RPPDS. The metabolite data of the embryos of PPDS and RPPDS were log transformed and

465 auto scaled for normalization, and then used for PCA analysis. The metabolite data of the

466 megagametophytes of PPDS and RPPDS were also subjected to PCA analysis after auto scaled for

467 normalization.

468 Partial least squares-discriminant analysis (PLS-DA) was conducted over 20 pairs of samples

469 (PPDS-0-E vs. PPDS-1-E, PPDS-1-E vs. PPDS-2-E, PPDS-2-E vs. PPDS-4-E, PPDS-4-E vs.

470 PPDS-6-E, PPDS-1-E vs. PPDS-4-E, PPDS-1-E vs. PPDS-6-E, PPDS-0-M vs. PPDS-1-M,

471 PPDS-1-M vs. PPDS-2-M, PPDS-2-M vs. PPDS-4-M, PPDS-4-M vs. PPDS-6-M, PPDS-1-M vs.

472 PPDS-4-M, PPDS-1-M vs. PPDS-6-M, RPPDS-0-E vs. RPPDS-1-E, RPPDS-1-E vs.

473 RPPDS-SCR-E, RPPDS-SCR-E vs. RPPDS-RP-E, RPPDS-0-M vs. RPPDS-1-M, RPPDS-1-M vs.

474 RPPDS-SCR-M, RPPDS-SCR-M vs. RPPDS-RP-M, PPDS-4-E vs. RPPDS-1-E, PPDS-4-M vs.

475 RPPDS-1-M, PPDS-4-E vs. RPPDS-SCR-E and PPDS-4-M vs. RPPDS-SCR-M) to determine

476 whose metabolites contribute significantly to the separation of two samples. Those metabolites

477 with VIP (variable importance in the projection) > 1 and significant changes (P < 0.05) in relative

478 contents were differentially expressed between two samples. The metabolite data were also

479 subjected to a combined normalization treatment of log transformation and auto scaling (or pareto

480 scaling alone) before performing PLS-DA.

481 The metabolites (VIP > 1 and P < 0.05) data between the embryos and megagametophytes of

482 PPDS (or RPPDS) were used to produce volcano plot. The volcano plot is a combination of fold

483 change and t-tests. The x-axis is log2(FC) in volcano plot. Fold change (FC) was calculated as the

484 ratio of the relative contents of metabolites in embryo to megagametophyte and log2 transformed.

485 FC threshold was set as 1.0. Y-axis is -log10 (p.value), and was based on raw p values. Log2(FC)

486 value greater than zero indicates that the relative contents of metabolites are higher in embryo than

487 megagametophyte. If log2(FC) value is less than zero, this suggest that the relative contents of

488 metabolites are lower in embryo compared with megagametophyte. Metabolic pathway analysis

489 was also performed with metabolites (VIP > 1 and P < 0.05) between any of 22 pairs of samples

490 using the MetaboAnalyst web tool to determine whose metabolic pathways are significantly

491 changed. The relative parameters were set using the same method to that used by Song and Zhu

492 (2019).

493 ACKNOWLEDGMENTS

494 We thank Xinghuan Li and Chunxiang Chen for their field support. This study was supported by

495 the National Natural Science Foundation of China (31901300, 31960636) and Natural Science

496 Foundation of Guizhou Province (2019) No. 1165.

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679

680 Table 1. The altered metabolic pathways in the embryo and megagametophyte of primary

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682 megagametophyte. “” indicates significantly changed metabolic pathway between two time

683 points.

Metabolic 0 week 1 week vs. 2 weeks vs. 4 weeks vs. 1 week vs. 1week vs. pathways vs. 1 week 2 weeks 4 weeks 6 weeks 4 weeks 6 weeks Em Me Em Me Em Me Em Me Em Me Em Me Carbohydrate Pentose phosphate        metabolism pathway C5-Branched dibasic        acid metabolism Fructose and mannose     metabolism Amino sugar and       nucleotide sugar metabolism Pentose and      glucuronate interconversions Glycolysis /  Gluconeogenesis Pyruvate metabolism  Glyoxylate and      dicarboxylate metabolism Starch and sucrose    metabolism Galactose metabolism   Citrate cycle (TCA      cycle) Butanoate metabolism     Carbon fixation in  photosynthetic organisms Inositol phosphate     metabolism Amino acid Alanine, aspartate and           metabolism glutamate metabolism 32

Arginine and proline      metabolism Arginine biosynthesis      Cysteine and      methionine metabolism Tryptophan   metabolism Tyrosine metabolism        Phenylalanine  metabolism Glycine, serine and   threonine metabolism Phenylalanine,   tyrosine and tryptophan biosynthesis Nucleotide Pyrimidine       metabolism metabolism Purine metabolism     Lipid Glycerophospholipid       metabolism metabolism Linoleic acid       metabolism Glycerolipid     metabolism Cutin, suberine and   wax biosynthesis Biosynthesis of Isoquinoline alkaloid        other secondary biosynthesis metabolites Biosynthesis of   other secondary metabolites-uncl assified Metabolism of Pantothenate and CoA     cofactors and biosynthesis vitamins Metabolism of Glutathione  other amino metabolism acids

684

685 Table 2. The altered metabolic pathways in the embryo and megagametophyte of seeds with

686 released primary physiological dormancy between successive time points. Em: embryo, Me:

687 megagametophyte. SCRS: seed coat rupture stage, RPS: radicle protrusion stage. “” indicates

688 significantly changed metabolic pathway between two time points.

689 Metabolic pathways 0 week 1 week vs. SCRS vs. vs. 1 week SCRS RPS Em Me Em Me Em Me Carbohydrate metabolism Pentose phosphate pathway      C5-Branched dibasic acid metabolism   Fructose and mannose metabolism      Amino sugar and nucleotide sugar     metabolism Pentose and glucuronate interconversions  Glycolysis / Gluconeogenesis Pyruvate metabolism Glyoxylate and dicarboxylate metabolism Starch and sucrose metabolism      Galactose metabolism     Citrate cycle (TCA cycle)  Butanoate metabolism     Carbon fixation in photosynthetic organisms Inositol phosphate metabolism     Ascorbate and aldarate metabolism     Amino acid metabolism Alanine, aspartate and glutamate metabolism       Arginine and proline metabolism    Arginine biosynthesis     Cysteine and methionine metabolism    Tryptophan metabolism     Tyrosine metabolism  Phenylalanine metabolism Glycine, serine and threonine metabolism     Phenylalanine, tyrosine and tryptophan biosynthesis Phenylalanine metabolism   Nucleotide metabolism Pyrimidine metabolism       Purine metabolism Lipid metabolism Glycerophospholipid metabolism    Linoleic acid metabolism   Glycerolipid metabolism   34

Cutin, suberine and wax biosynthesis Biosynthesis of other Isoquinoline alkaloid biosynthesis  secondary metabolites Biosynthesis of other    secondary metabolites-unclassified Metabolism of cofactors Pantothenate and CoA biosynthesis   and vitamins Metabolism of other Glutathione metabolism     amino acids

690

691

692

693

694

695

696

697

698

699

700

701

702

703

704

705

706

707 Table 3. The altered metabolic pathways in the embryo and megagametophyte between

708 4-weeks-incuabted primary physiological dormant seeds (PPDS-4) and 1-weeks-incubated seeds

709 with released primary physiological dormancy (RPPDS-1), between PPDS-4 and seeds with

710 released primary physiological dormancy at seed coat rupture stage (RPPDS-SCR). Em: embryo,

711 Me: megagametophyte. “” indicates significantly changed metabolic pathway.

712 Metabolic pathways PPDS-4 PPDS-4 vs. vs. RPPDS-1 RPPDS-SCR Em Me Em Me Carbohydrate metabolism Pentose phosphate pathway     C5-Branched dibasic acid metabolism  Fructose and mannose metabolism     Amino sugar and nucleotide sugar metabolism    Pentose and glucuronate interconversions    Glycolysis / Gluconeogenesis Pyruvate metabolism Glyoxylate and dicarboxylate metabolism  Starch and sucrose metabolism    Galactose metabolism     Citrate cycle (TCA cycle)    Butanoate metabolism    Carbon fixation in photosynthetic organisms Inositol phosphate metabolism     Ascorbate and aldarate metabolism  Amino acid metabolism Alanine, aspartate and glutamate metabolism     Arginine and proline metabolism     Arginine biosynthesis    Cysteine and methionine metabolism  Tryptophan metabolism  Tyrosine metabolism    Phenylalanine metabolism Glycine, serine and threonine metabolism    Phenylalanine, tyrosine and tryptophan  biosynthesis Phenylalanine metabolism Nucleotide metabolism Pyrimidine metabolism   Purine metabolism   Lipid metabolism Glycerophospholipid metabolism Linoleic acid metabolism  36

Glycerolipid metabolism  Cutin, suberine and wax biosynthesis Biosynthesis of other Isoquinoline alkaloid biosynthesis     secondary metabolites Biosynthesis of other  secondary metabolites-unclassified Metabolism of cofactors and Pantothenate and CoA biosynthesis   vitamins Metabolism of other amino Glutathione metabolism    acids

713

714

715

716

717

718

719

720

721

722

723

724

725

726

727

728

729 Figure captions

730 Figure 1. Principal components analysis (PCA) of metabolite profiles of (A) the embryos of five

731 primary physiological dormancy seeds (PPDS) samples (PPDS-0-E, PPDS-1-E, PPDS-2-E,

732 PPDS-4-E and PPDS-6-E) and the megagametophytes of five PPDS samples (PPDS-0-M,

733 PPDS-1-M, PPDS-2-M, PPDS-4-M and PPDS-6-M), (B) the embryos of four seeds with released

734 primary physiological dormancy (RPPDS) samples (RPPDS-0-E, RPPDS-1-E, RPPDS-SCR-E

735 and RPPDS-RP-E) and the megagametophytes of four RPPDS samples (RPPDS-0-M,

736 RPPDS-1-M, RPPDS-SCR-M and RPPDS-RP-M), (C) the embryos of PPDS (PPDS-0-E,

737 PPDS-1-E, PPDS-2-E, PPDS-4-E and PPDS-6-E) and RPPDS (RPPDS-0-E, RPPDS-1-E,

738 RPPDS-SCR-E and RPPDS-RP-E), and (D) the megagametophytes of PPDS (PPDS-0-M,

739 PPDS-1-M, PPDS-2-M, PPDS-4-M and PPDS-6-M) and RPPDS (RPPDS-0-M, RPPDS-1-M,

740 RPPDS-SCR-M and RPPDS-RP-M).

741 Figure 2. Fold change in the relative contents of important metabolites with VIP value > 1 and P

742 < 0.05 in the embryos of primary physiological dormancy seeds (PPDS) (A) between 0 and 1

743 week of incubation (PPDS-0-E vs. PPDS-1-E), (B) 1 and 2 weeks of incubation (PPDS-1-E vs.

744 PPDS-2-E), (C) between 2 and 4 weeks of incubation (PPDS-2-E vs. PPDS-4-E), and (D) 4 and 6

745 weeks of incubation (PPDS-4-E vs. PPDS-6-E), (E) between 1 and 4 weeks of incubation

746 (PPDS-1-E vs. PPDS-4-E), and (F) 1 and 6 weeks of incubation (PPDS-1-E vs. PPDS-6-E).

747 Figure 3. Volcano plots of the important metabolites with VIP value > 1 and P < 0.05 between

748 embryo and megagametophyte of primary physiological dormancy seeds at five different

749 incubation time points.

750 Figure 4. Fold change in the relative contents of important metabolites with VIP value > 1 and P

751 < 0.05 in the megagametophytes of primary physiological dormancy seeds (PPDS) (A) between 0

752 and 1 week of incubation (PPDS-0-M vs. PPDS-1-M), (B) 1 and 2 weeks of incubation

753 (PPDS-1-M vs. PPDS-2-M), (C) between 2 and 4 weeks of incubation (PPDS-2-M vs.

754 PPDS-4-M), (D) 4 and 6 weeks of incubation (PPDS-4-M vs. PPDS-6-M), (E) between 1 and 4

755 weeks of incubation (PPDS-1-M vs. PPDS-4-M), and (F) 1 and 6 weeks of incubation (PPDS-1-M

756 vs. PPDS-6-M).

757 Figure 5. Fold change in the relative contents of important metabolites with VIP value > 1 and P

758 < 0.05 in the embryo of seeds with released primary physiological dormancy seeds (RPPDS) (A)

759 between 0 and 1 week of incubation (RPPDS-0-E vs. RPPDS-1-E), and (B) 1 week of incubation

760 and seed coat rupture stage (RPPDS-1-E vs. RPPDS-SCR-E), and (C) seed coat rupture stage and

761 radicle protrusion stage (RPPDS-SCR-E vs. RPPDS-RP-E).

762 Figure 6. Volcano plots of the important metabolites with VIP value > 1 and P < 0.05 between

763 embryo and megagametophyte of seeds with release primary physiological dormancy at four

764 incubation time points.

765 Figure 7. Fold change in the relative contents of important metabolites with VIP value > 1 and P

766 < 0.05 in the megagametophyte of seeds with released primary physiological dormancy seeds

767 (RPPDS) (A) between 0 and 1 week of incubation (RPPDS-0-M vs. RPPDS-1-M), and (B) 1 week

768 of incubation and seed coat rupture stage (RPPDS-1-M vs. RPPDS-SCR-M), and (C) seed coat

769 rupture stage and radicle protrusion stage (RPPDS-SCR-M vs. RPPDS-RP-M).

770 Figure 8. Fold change in the relative contents of important metabolites with VIP value > 1 and P

771 < 0.05 in the embryo of (A) between seeds with released primary physiological dormancy seeds

772 (RPPDS) incubated for 1 week (RPPDS-1-E) and primary physiological dormancy seeds (PPDS)

773 incubated for 4 weeks (PPDS-4-E), (B) between RPPDS with seed coat rupture (RPPDS-SCR-E)

774 and PPDS-4-E, in the megagametophytes of (C) between seeds with released primary

775 physiological dormancy seeds (RPPDS) incubated for 1 week (RPPDS-1-M) and primary

776 physiological dormancy seeds (PPDS) incubated for 4 weeks (PPDS-4-M), and (D) between

777 RPPDS with seed coat rupture (RPPDS-SCR-M) and PPDS-4-M.

778 779

40 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.08.430286; this version posted February 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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