Putative Spatiotemporal Changes in Inhibitor Activity during Cold Stratification of Sapium sebiferum Seeds

Abstract

Sapium sebiferum is a tree with socioeconomic, environmental, and medicinal value. S. sebiferum seeds possess physiological dormancy, which is induced by endogenous inhibitors and can be broken by cold stratification. However, the putative spatiotemporal changes in inhibitor activity are currently unknown, which can hinder the propagation of S. sebiferum seeds. The objective of this study was to investigate the spatiotemporal dynamics of inhibitor activity and its effect on germination during the cold stratification of S. sebiferum seeds. An extractant consisting of 80% methanol was used to extract the inhibitory substance from the seed coat and endosperm at different stages of cold stratification. The extract was then applied to both the Chinese cabbage seeds and excised embryos of S. sebiferum. The germination percentage and germination index were used to assess the inhibitor activity of S. sebiferum seeds. The germination of non-stratified S. sebiferum seeds was completely inhibited (0% germination). As the stratification duration was extended, the germination percentage of S. sebiferum seeds gradually increased. However, dormancy persisted until the stratification duration reached 120 d; at this point, the mean germination was 81.3%. The germination test on Chinese cabbage seeds revealed a significant increase from 10.0% (stratified for 0 d) to 91.2% (stratified for 120 d) when treated with endosperm extracts. The germination indexes also increased from 0.0 to 40.3, indicating a decrease in the inhibitory activity of endosperm extracts. The seed coat extracting solution showed varying dynamic changes. The lowest germination was observed after 60 d of stratification, with no significant differences among the results of 0 d, 30 d, and 60 d. However, after stratification for 90 d, the germination percentage of Chinese cabbage seeds increased. The germination percentage of excised embryos exhibited similar changes to those of Chinese cabbage seeds. This study discovered that endogenous inhibitors were present in both the seed coat and endosperm of S. sebiferum seeds, and the inhibitor activity was higher in the endosperm. The spatiotemporal patterns of inhibitor activity suggest that the endogenous inhibitors move from the endosperm to the seed coat during early cold stratification stages, aiding in the physiological dormancy release of S. sebiferum seeds. These findings enhance our understanding of seed biology in S. sebiferum and will facilitate high-efficiency seed propagation.

1. Introduction

Seed dormancy is one of the most important adaptive traits formulated during the evolution of plants, especially in perennial species, which could help to improve the ability of seeds to withstand adversity and ensure the continuity of genetic information [1,2]. Dormancy also plays a crucial role in expanding the geographical distribution of the species and increasing population dominance [2,3,4]. However, dormancy can severely limit seedling production. Seed dormancy can be induced by several factors. The internationally accepted classification system for dormancy, proposed by Baskin and Baskin [2], includes five classes: physical, combinational, physiological, morphological, and morphophysiological. Physiological dormancy is typically caused by endogenous inhibitors in the seeds that can inhibit or delay the physiological metabolic activity of the seed, thereby affecting seed germination. This phenomenon has been widely observed in various plant species, such as Quercus aliena [5], Bunium persicum [6], Trillium kamtschaticum [7], Grevillea petrophiloides [8], and Fritillaria taipaiensis [9]. As a common method of breaking seed dormancy, cold stratification could induce a temporal decrease in the concentration of germination inhibitors in Taxus yunnanensis seeds [10]. Similarly, Gao et al. [11] observed a gradual decrease in the activity of endogenous inhibitors in Cercus chinensis seeds with the extension of the stratification duration, which ultimately broke the dormancy.

Sapium sebiferum is one of the most widely planted tree species in the Euphorbiaceae family and has been cultivated in China for over 1400 years. S. sebiferum is renowned for its colorful leaves in autumn, transitioning from green to gorgeous yellow or red. Additionally, it holds significant economic value as a crucial raw material source for biodiesel production [12]. The white waxy aril of the seeds can be extracted as a raw material for making candles and soap and in paint manufacturing, while the catalpa oil in the seeds can be used in paints, inks, and cosmetics [13,14,15]. In addition, S. sebiferum has high medicinal value as its leaves and roots are used in traditional Chinese medicine. Due to its great ornamental, economic, and medicinal value, S. sebiferum has gained increasing attention from the Chinese government and has become a focus of cultivation throughout China.

Nowadays, S. sebiferum is mainly propagated from harvested seeds, but seed dormancy is a major challenge for seedling propagation. Previous research has shown that the seed dormancy of S. sebiferum was mainly induced by inhibitors [15,16], and the inhibitory levels in the endosperm have been proven to be stronger than those in the seed coat, regardless of their origin [17]. As expected, cold stratification could effectively release the dormancy of S. sebiferum seeds [16]. However, the temporal–spatial variation of inhibitors in S. sebiferum seeds during cold stratification remains largely unknown. In this study, mature S. sebiferum seeds were used as materials, with methanol as the extractant to extract inhibitors from the seed coat and endosperm of S. sebiferum seeds subjected to different durations of stratification. The germination percentage of Chinese cabbage seeds treated with different extractants was used to compare the dynamic changes in the concentration of germination inhibitors. This study aims to provide insights into the physiological changes in S. sebiferum seeds during the stratification process and the correlation between inhibitor content and seed dormancy. These findings will contribute to help the scientific basis for S. sebiferum seedling production.

2. Materials and Methods

2.1. Seed Materials

S. sebiferum seeds were collected in November 2020 from one tree in Xuanwu Lake Park, Nanjing, Jiangsu Province. The geographic location is 32°4′16″ north latitude and 118°48′10″ east longitude. Firstly, we soaked the seeds in a 1% sodium hydroxide solution for 20 min to remove the white waxy tallow completely. Then, the seeds were naturally dried and stored in sealed plastic bags in a refrigerator at 4 °C until the related experiments. The commercial Chinese cabbage seeds (Brassica rapa) with purity ≥ 95.0% and germination (%) ≥ 85.0% were used for the experiment.

2.2. Cold Stratification

The S. sebiferum seeds (about 3000 seeds) were soaked in water at room temperature for 72 h, with the water being changed every 24 h. After that, the seeds (about 600 seeds per treatment) were stratified at 4 °C in moist sand at a volume ratio of 1:3 for 0, 30, 60, 90, and 120 d, respectively. The sand’s humidity should allow it to be formed into a ball when held in the hand and then easily dispersed when loosened. During stratification, the seeds were turned regularly to maintain good aeration and watered to ensure adequate humidity for effective stratification. After each stratification period, the seeds were divided into two parts for subsequent experiments: one part for the seed germination test and the other for solvent extraction.

2.3. Germination Tests of S. sebiferum Seeds after Different Stratification Periods

After each stage of cold stratification, seed germination tests were conducted using 30 seeds with three replicates for each test. The tests were carried out in a growth chamber at a constant temperature of 25 °C, with altering light exposure of 8 h of light followed by 16 h of darkness. Germination was counted every other day for 30 d. Thirty days later, the germination percentages were determined according to the Rules of ISTA [18].

Following the germination tests, the viability of the ungerminated seeds was assessed using the tetrazolium (TZ) test according to the Rules of ISTA [18]. The TZ test procedure involved removing the seed coat of non-germinated seeds and staining the remaining parts in a 1.0% (w/v) solution of 2,3,5-triphenyl-tetrazolium chloride (TTC) at 35 °C in the dark for 6 h. Seeds were judged as viable when they showed a completely stained embryo and most parts of the endosperm (small, unstained areas in the endosperm can be accepted). Seed viability was recorded and calculated according to ISTA [18].

2.4. Effects of Extracted Solvents on Chinese Cabbage Seeds

Before examining the impact of the extraction solvent on the germination of S. sebiferum seeds at different stratification stages, we investigated whether the extractant itself had an effect on the germination of the Chinese cabbage seeds. The extracted solvents were distilled at 60 °C to eliminate the methanol in the solution. The Chinese cabbage seeds were treated with 5 mL of distilled residue for 3 h, while the control group was soaked in deionized water for the same duration. Then, the seeds were placed in a 10 mL beaker with three replicates of 100 seeds each. After immersion, they were transferred to glass Petri dishes (about 10 cm diameter) with two pieces of filter paper (about 9 cm diameter) at the bottom. The dishes were then incubated for germination tests at 25 °C, with an 8 h light and 16 h darkness. The number of germinated seeds, defined as those with a radicle elongated to approximately 1 cm, was recorded every 8 h for each treatment. Germination was considered complete when the percentage of germination per day did not exceed 1% of repeated particles for 3 consecutive days [19]. Germination percentages were calculated as described above, and the germination indexes (GIs) were calculated as follows: GI = ∑(Gt/Dt). Gt represents the number of germinated seeds at different times (t), and Dt is the corresponding number of days to germination [20].

2.5. Germination Tests of Seed Extracts from Dynamic Stratification Stages of Seed Coat and Endosperm on Chinese Cabbage Seeds

In this experiment, seed extracts of S. sebiferum were obtained by methanol extraction as in the procedure described by Zhao et al. [20]. First, 5 g of seed coat and endosperm of S. sebiferum seeds (about 200 stratified seeds) with different stratification periods was separated. The seed coat and the endosperm were ground with a mortar. The ground powder was homogenized in a 40 mL solution of 80% (v/v) methanol in a refrigerator at 5 °C for 24 h and filtered in a 15 cm diameter Ederol No. 11 filter paper. Then, the filtered residues were re-extracted twice with 40 mL of 80% (v/v) methanol as described above. Finally, the combined methanol filtrates were evaporated under a vacuum at 60 °C to expulse methanol. The final volume was adjusted to 20 mL with distilled water (concentration~0.25 g·mL⁻¹).

Germination tests of Chinese cabbage seeds were conducted to determine the inhibitory effects of seed coat and endosperm extracts after dynamic stages of stratification. The extracted solution (5 mL) was added to the Chinese cabbage seeds for 3 h in a 10 mL beaker, while the control group was treated with distilled water for the same period. The Chinese cabbage seeds with three replicates of 100 seeds each were treated. Throughout the germination period, the test solution or water was added promptly to the filter paper (about 9 cm diameter) to maintain the humidity. The germination procedures were carried out as described above.

2.6. Effects of Seed Extracts on the Germination of Isolated S. sebiferum Embryos

Mature S. sebiferum seeds were soaked in running water for 2 d. After excision from their enclosing seed tissues, the embryos were placed in glass Petri dishes (about 10 cm diameter) containing two pieces of filter paper (about 9 cm diameter) moistened with each of the extract solutions described above and incubated at 25 °C with an 8 h light and 16 h darkness period. The germination tests of the excised embryos were conducted using three replicates with 30 seeds each. Germination was monitored every other day for 16 d. Finally, the germination percentages and germination indexes of the excised embryos were calculated.

2.7. Statistical Analysis

Data on the germination percentage and germination index from each experiment were analyzed separately. Significance was tested by one-way analysis of variance (ANOVA) and Duncan’s multiple range tests were used to identify significant differences between pairs of means at p ≤ 0.05.

3. Results

3.1. Dynamic Changes in Germination Percentage of S. sebiferum Seeds at Different Cold Stratification Periods

The germination percentage and seed viability of S. sebiferum seeds stratified for various periods are shown in

Table 1. Effects of different cold stratification periods on the germination of Sapium sebiferum seeds.

Stratification Period (d)	Germination Percentage (%) †	Seed Viability of the Ungerminated Seeds (%) ††
0	0.0 ± 0.0 d	85.0
30	2.0 ± 0.0 d	79.2
60	16.7 ± 2.4 c	78.6
90	40.0 ± 4.0 b	67.4
120	81.3 ± 1.7 a	3.3

^† Each value is a mean (n = 30 × 3) ± standard deviation (SD). Values within a column followed by different lower-case letters are significantly different at p ≤ 0.05 by Duncan’s test, and the same is below. ^†† Seed viability of the remaining seeds without germination (%) = $\frac{N_{\mathrm{Staining}}}{N_{\mathrm{Ungerminated}}}$ × 100.

. Seeds stratified for 0 d did not germinate at all. The viability of ungerminated seeds stained by TTC was 85.0%, indicating that most of the ungerminated seeds had germination potential and the reason for their non-germination was the failure to break dormancy. As the stratification duration was extended, the germination percentage of S. sebiferum seeds increased significantly, while the viabilities of the ungerminated seeds decreased rapidly. When S. sebiferum seeds were stratified for 90 d, the germination percentage significantly increased to 40.0%, and the viability of ungerminated seeds decreased to 67.4%. Therefore, even after a stratification of 90 d, 67.4% of viable S. sebiferum seeds were still unable to germinate successfully. After 120 d of stratification, nearly all the viable seeds had germinated, with the germination percentage of S. sebiferum seeds reaching its peak at 81.3%. These results provide valuable insights into the fact that cold stratification gradually breaks seed dormancy and promotes seed germination.

3.2. Effect of Extraction Solvent on the Germination of Chinese Cabbage Seeds

Before examining the effects of S. sebiferum seed extracts on the germination of Chinese cabbage seeds, we first conducted a test to determine whether the extract itself would have an impact on the results (

Table 2. Effect of extraction solvent on the germination of Chinese cabbage seeds.

Treatment	Germination Percentage (%)	Germination Index
Control	92.3 ± 0.9 a	43.8 ± 0.4 a
80% (v/v) methanol solution	93.7 ± 1.2 a	45.2 ± 1.3 a

Values within a column followed by different lower-case letters are significantly different at p ≤ 0.05 by Duncan’s test.

). As Table 2 shows, the treatment’s germination percentage and germination index were not significantly different from those of the control. Thus, in this experiment, the extraction solvent could be completely evaporated by distillation without affecting the germination of Chinese cabbage seeds.

3.3. Effects of Endosperm Extracts on the Germination of Chinese Cabbage Seeds

Table 3. Effects of inhibitors extracted from the endosperm of Sapium sebiferum seeds stratified for different periods on the germination ability of Chinese cabbage seeds.

Stratification Period (d)	Germination Percentage (%)	Germination Index
Control	92.3 ± 0.9 a	43.8 ± 0.4 a
0	10.0 ± 4.0 d	0.0 ± 0.0 d
30	67.0 ± 2.0 c	9.2 ± 0.4 c
60	78.7 ± 1.0 bc	9.8 ± 1.0 c
90	83.7 ± 4.2 b	23.0 ± 4.5 b
120	91.2 ± 1.4 a	40.3 ± 2.1 a

Values within a column followed by different lower-case letters are significantly different at p ≤ 0.05 by Duncan’s test.

provides the experimental results of the endosperm extracts on the germination of Chinese cabbage seeds. When Chinese cabbage seeds were treated with the endosperm extracts of S. sebiferum seeds without stratification (0 d), the germination percentage was only 10.0%, strongly lower than that of the control (92.3%), when treated with distilled water. Also, the most striking result of the table was the GI value of the endosperm extracts from the non-stratified group (0 d), which was 0, because all the Chinese cabbage seeds germinated after 88 h and it caused the most significant decline in the GI value (100.0%). As the stratification duration extended to 30 d, the germination percentage of the Chinese cabbage seeds treated with corresponding endosperm extracts increased sharply to 67.0% compared with 10% without stratification, and a slower increase in GI value occurred to 9.2. When S. sebiferum seeds were stratified for 90 d, the germination percentage of Chinese cabbage seeds increased to 83.7%, and the GI value increased greatly to 23.0. Although the germination percentage reached a relatively high level, it was still significantly lower than that of the control. Therefore, for S. sebiferum seeds, stratification for 90 d still could not completely break the seed dormancy, so when the stratification duration of S. sebiferum seeds further extended to 120 d, the germination percentage and GI value increased further to 91.2% and 40.3, respectively, which were not significantly different from those of the control. In general, as the stratification duration extended, both the germination percentage and GI values showed increasing trends. Taken together, these results demonstrated the endosperm extracts did contain germination inhibitors and truly affected the germination of Chinese cabbage seeds. Furthermore, the longer the duration of the stratification treatment, the greater the decrease in inhibitory activity.

3.4. Effects of Seed Coat Extracts of S. sebiferum on the Germination of Chinese Cabbage Seeds

The results of seed coat extracts on the germination percentages and GI values of Chinese cabbage seeds are summarized in

Table 4. Effects of inhibitors extracted from Sapium sebiferum seed coats after different stratification periods on the germination of Chinese cabbage seeds.

Stratification Period (d)	Germination Percentage (%)	Germination Index
Control	92.3 ± 0.9 a †	43.8 ± 0.4 a
0	80.3 ± 0.9 b	22.0 ± 2.0 c
30	80.3 ± 3.3 b	11.3 ± 2.8 d
60	79.3 ± 4.8 b	16.3 ± 1.4 d
90	88.7 ± 1.8 ab	31.6 ± 1.2 b
120	90.8 ± 0.9 a	41.6 ± 0.9 a

^† Each value is a mean (n = 30 × 3) ± standard deviation (SD). Values within a column followed by different lower-case letters are significantly different at p ≤ 0.05 by Duncan’s test.

. The germination percentage of the seed coat showed a quite different trend compared to that of the endosperm, with a decrease followed by an increase. Compared to the control, the germination percentage of Chinese cabbage seeds was significantly reduced by the seed coat extracts stratified for 0, 30, and 60 d. However, no significant difference was found among the three treatments. For S. sebiferum seeds stratified for 90 and 120 d, no significant differences were found between the treatments and the control. This meant that when the stratification duration was prolonged enough, the inhibitory activities on germination were weakened or even vanished.

The initial GI of Chinese cabbage seeds was 22.0 ± 2.0 after treatment with newly collected S. sebiferum seed coat extracts. However, when treated with extracts of seed coats stratified for 30 d, the GI decreased significantly to 11.3 ± 2.8. As the stratification duration extended from 30 to 120 d, the GI gradually increased from 11.3 ± 2.8 to 41.6 ± 0.9. The results of the ANOVA analysis indicated that the GI values of Chinese cabbage seeds that were treated with S. sebiferum seed coat extracts stratified for 0–90 d were significantly lower than those of the control group. However, when the seeds were stratified for 120 d, the seed coat extracts did not have a significant effect on the GI value compared to the control. In summary, the changes in germination percentage and GI value indicated that the inhibitors of germination did exist in the S. sebiferum seed coat of each stratification stage except for the seeds stratified for 120 d, which meant cold stratification could effectively break seed dormancy and promote the germination of S. sebiferum seeds.

3.5. Effects of Extracts on the Germination of Excised Embryos of S. sebiferum Seeds

Apart from germination tests on Chinese cabbage seeds, we also examined the effects of S. sebiferum seed coat and endosperm extracts on the germination of excised S. sebiferum embryos. As the stratification duration was extended, the germination percentage of excised embryos treated with endosperm extracts displayed a sharply increasing trend. The germination percentage of S. sebiferum excised embryos cultured with distilled water (control) was 84.7%, while that of the excised embryos treated with non-stratified seed endosperm extracts dramatically decreased to 11.2%, and the germination index decreased by 95.5%. These results demonstrated that the inhibitors of S. sebiferum seed endosperm extracts could significantly repress the germination of excised embryos. In addition, with the prolongation of the cold stratification duration, the germination percentage of excised embryos gradually increased. This indicated that the activity of inhibitors in endosperm declined during the stratification process. When the excised embryos were treated with the endosperm extracts stratified for 120 d, the germination percentage and the GI value of the treatment were not significantly different from those of the control, which was consistent with the previous bioassay of Chinese cabbage seeds treated with endosperm extracts.

Meanwhile, the changing trend of the germination percentage of excised embryos was approximately 62% when treated with seed coat extracts stratified for 0–60 d, but increased dramatically to 82.7 ± 2.1 for extracts stratified for 120 d. In contrast, GI values gradually increased, similar to those treated with endosperm. When compared with the control, the germination percentage of excised embryos was reduced by the seed coat extracts stratified for 0, 30, and 60 d. However, there was no significant difference among the three treatments, which were all higher than the endosperm extract treated for the same time. Consistent with the results of previous germination tests on Chinese cabbage seeds, when the excised embryos were treated with the seed coat extracts stratified for 90 d and more, there were no significant differences between the treatments and the control, which meant that the inhibitory activities on germination would be weakened significantly after 90 d of stratification.

4. Discussion

It is widely accepted that temperature regulates dormancy. Previous studies have demonstrated that cold stratification can effectively break seed dormancy by inducing a temporal decrease in the activity of endogenous inhibitors [1,2,21]. For example, the seeds of Fagus sylvatica and Taxus yunnanensis showed the inhibitors declined during the process of seed dormancy release [10]. Likewise, the study of inhibitor alterations in Cercis chinensis seeds revealed a decline in inhibitors during the process of seed dormancy release [11]. The results of our research indicate that the germination percentages of Chinese cabbage seeds gradually increased when exposed to extracts of inhibitors from the seed coat and endosperm with different durations of cold stratification (Table 3 and Table 4). So, the activity of endogenous inhibitors temporally decreased during cold stratification. In addition, seed coat and endosperm extracts inhibit the germination of Chinese cabbage seeds, but do the inhibitory effects affect the germination of S. sebiferum embryos? To test this, we conducted a repeated experiment using excised embryos from S. sebiferum seeds instead of Chinese cabbage seeds and obtained consistent results (

Table 5. Effects of inhibitors extracted from the stratified Sapium sebiferum seeds on the germination of excised embryos.

Position	Stratification Period (d)	Germination Percentage (%)	Germination Index
Control		84.7 ± 6.7 a	6.7 ± 0.1 a
Endosperm	0	11.2 ± 2.3 d	0.3 ± 0.0 d
	30	42.3 ± 5.0 c	3.2 ± 0.2 c
	60	59.3 ± 2.7 bc	3.8 ± 0.1 c
	90	78.6 ± 3.2 b	5.6 ± 0.1 b
	120	83.1 ± 2.2 a	6.3 ± 0.1 a
Seed coat	0	62.1 ± 3.1 b	3.4 ± 0.3 c
	30	62.0 ± 6.3 b	3.9 ± 0.2 c
	60	61.3 ± 5.6 b	4.3 ± 0.2 bc
	90	80.7 ± 2.9 a	5.9 ± 0.3 ab
	120	82.7 ± 2.1 a	6.4 ± 0.3 a

Values within a column followed by different lower-case letters are significantly different at p ≤ 0.05 by Duncan’s test.

).

Endogenous inhibitors are species-specific and are present in various parts of seeds, such as the pericarp, seed coat, endosperm, and embryo [2]. In T. yunnanensis and T. henryana seeds, inhibitors induced physiological dormancy, and their activity varied between the seed coat and endosperm [10,22]. In C. chinensis seeds, the seed coat exhibited higher inhibitor activity than the endosperm [11]. For S. sebiferum seeds, the initial activity of inhibitors from the endosperm was much higher than that from the seed coat, as demonstrated by germination tests (Table 3, Table 4 and Table 5), which was consistent with the previous results [16,17]. Cold stratification is a common method to reduce the activity or concentration of endogenous inhibitors through degradation or transport [2]. According to Mousavi et al. [23], many inhibitors can be leached from the seed, thus shifting the balance towards the growth-promoting chemicals and allowing them to germinate. Obviously, the endogenous inhibitors exhibited spatiotemporal dynamic changes to a certain degree. In this study, the activity of inhibitors from the endosperm and seed coat showed different change patterns with the extension of the cold stratification duration. The inhibition ability of endosperm inhibitors continued to decrease during cold stratification (Table 3), while the seed coat primarily maintained high levels and then declined after stratification for 90 d (Table 4). The extract from both the seed coat and endosperm exhibited the same results in the germination of excised embryos (Table 5). This phenomenon was quite similar to the observation on Taxus yunnanensis seeds, and the underlying cause is that the inhibitors might be transferred between the seed coat and the kernel during the cold stratification [10]. Mousavi et al. [23] considered that for physiological dormancy release, the endosperm must be metabolically active and the seed coat must be at least semi-permeable to water to allow respiration inhibitors to leach out of the seed. Therefore, it is hypothesized that endogenous inhibitors are transferred from the endosperm to the seed coat in S. sebiferum during cold stratification for 0–60 d. Zhao et al. identified 4, 14, 20, and 9 compounds in ether, methanol, ethyl acetate, and petroleum ether extracts, respectively, in their study of S. sebiferum inhibitors in four distinct fractions [20]. In addition, Shah et al. [15] pointed out that proanthocyanidins was the inhibitor for S. sebiferum seeds. However, they did not determine the specific content of these substances in various seed parts. Further studies are necessary to investigate the changes in these substances during the cold stratification of S. sebiferum seeds.

5. Conclusions

In conclusion, this study showed that the activity of inhibitors in S. sebiferum seeds underwent spatiotemporal changes during cold stratification. As the cold stratification duration was extended, the activity of the inhibitors decreased significantly over time. In addition, the pattern of inhibitors in different parts of the seeds varied. Inhibitor activity in the seed coat increased slightly during the initial 60 d of cold stratification and then decreased, while inhibitor activity in the endosperm continued to decrease. Therefore, the increase in inhibitor activity in the seed coat might be due to the outward movement of the inhibitor from the endosperm. Spatial metabolomics has matured and shows promise in the field of seed biology. The application of spatial metabolomics in studying seed dormancy release enables a thorough investigation of the altered regulation of inhibitors in different parts of the seed. Nevertheless, our findings primarily unveiled the putative spatiotemporal dynamics of inhibitor activity in S. sebiferum seeds during cold stratification and will serve as a theoretical basis for further high-efficiency seed propagation.