Exogenous gibberellic acid shortening after-ripening process and promoting seed germination in a medicinal plant Panax notoginseng

doi:10.21203/rs.3.rs-2111983/v1

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Research Article

Exogenous gibberellic acid shortening after-ripening process and promoting seed germination in a medicinal plant Panax notoginseng

https://doi.org/10.21203/rs.3.rs-2111983/v1

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published 01 Feb, 2023

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Background

Panax notoginseng (Burk) F.H. Chen is an important medicinal plant in the family of Araliaceae. Its seeds are classified as the type of morphophysiological dormancy (MPD), and are characterized by recalcitrance during the after-ripening process. However, it is not clear about the molecular mechanism on the dormancy in harvested recalcitrant seeds.

Results

In this study, exogenous supply of gibberellic acid (GA₃) with different concentrations shortened after-ripening process and promoted germination of P. notoginseng seeds. Among the identified plant hormone metabolites, exogenous GA₃ results in an increased levels of endogenous hormone GA₃ through permeation. A total of 2971 and 9827 differentially expressed genes (DEGs) were identified in response to 50 mg L^− 1 GA₃ (LG) and 500 mg L^− 1 GA₃ (HG) treatment, respectively, and the plant hormone signal and related metabolic pathways regulated by GA₃ was significantly enriched. GA₃ treatment upregulated the expression of ent-copalyl diphosphate synthase (CPS), GA 20-oxidase (GA20ox) and GIBBERELLIN INSENSITIVE DWARF1 (GID1), whereas it downregulated DELLA, Pyrabactin resistance 1-like (PYL) and ABA-INSENSITIVE5 (ABI5). This effect was associated with higher expression of crucial seed embryo development and cell wall loosening genes, Leafy Contyledon1 (LEC1), Late Embryogenesis Abundant (LEA), expansins (EXP) and Pectinesterase (PME).

Conclusions

Exogenous GA₃ application promotes the germination of P. notoginseng seeds by increasing GA₃ contents through permeation. Furthermore, the altered ratio of GA and ABA contributes to the development of the embryo, breaks the mechanical constraints of the seed coat and promotes the protrusion of the radicle in recalcitrant P. notoginseng seeds. These findings improve our knowledge of the contribution of GA to regulating dormancy of MPD seeds during the after-ripening process, and provide a new theoretical guidance for the application of recalcitrant seeds in agricultural production and storage.

Dormancy

Recalcitrant seeds

Gibberellic acid

After-ripening process

Panax notoginseng

The seed is the most critical developmental period in the life cycle of a plant [1]. Seed dormancy is traditionally defined as an intrinsic obstacle to germination under a favorable conditions [2–4], it is crucial for the conservation of germplasm resources, and for the prevention of pre-harvest sprouting [5]. Seed dormancy is determined by the morphological and physiological characteristics [6], and it is classified as physiological (PD), morphological (MD), physical (PY), combinational (PY + PD) and morpho-physiological (MPD) types [7, 8]. The MPD seeds have to undergo an after-ripening (AR) process characterized by a gradual reduction in dormancy level [9]. The seeds with the after-ripening process would undergo an intricate range of metabolic processes before germination, which is in preparation for the mobilization of food reserve and cell growth [10]. The after-ripening process depends on moisture and oil contents, seed covering structures and temperature [11]. With a low level of dormancy, temperatures and light could overcome the final limitations on germination, and the germination happens if water potential is adequate to allow the radicle to protrude [12]. The after-ripening is a process through which the dormancy is lost gradually with the increased duration of seed storage in a low hydrated state. However, it remains largely unknown about the mechanism of dormancy release of MPD seeds that show a high water content at the postharvest after-ripening process.

The elicitation, retention and reduction of seed dormancy is a highly intricate physiological process that relies on a multitude of endogenous and environmental factors [13]. Signals from hormones, essentially those of abscisic acid (ABA) and gibberellin (GA) are integrators between environmental cues and molecular signals, thus regulating gene expression [9, 14]. The ratio of GA and ABA is a critical factor to control seed dormancy and germination [4]. Fluctuating temperatures enhance the ratio of GA/ABA by decreasing ABA content in Cynara cardunculus seeds, and simultaneously the expression of Nine-cis-epoxycarotenoid dioxygenase (NCED) and ABA-INSENSITIVE5 (ABI5) is inhibited [15]. In the Arabidopsis and tomato (Lycopersicon esculentum M.), the mutants with defects in the gene encoding GA biosynthetic enzymes are unable to germinate [16] Pimenta Lange and Lange, 2006). The dormancy loss in wheat seeds is accompanied by the increased expression of TaGA20ox and with the enhanced level of bioactive GA₁ during imbibition [17, 18]. Arabidopsis plants constitutively expressing GA catalytic enzyme GA 2-oxidase (GA2ox) reveal that the reduced accumulation of GA in seeds leads to the increased probability of seed abortion [19]. The seed germination is dependent on gibberellin (GA) and is inhibited by DELLA when GA concentration is relatively low in Arabidopsis (Cao et al., 2006; Sun and Gubler, 2004). Seeds of GA-deficient mutant ga1 (GA Requiring 1) exhibit a failure to germinate phenotype in the lack of exogenous gibberellic acid (GA₃) [20]. In contrast, the mutant defective in GA2ox could deactivate bioactive GA, consequently accompanying with a decreased level of seed dormancy [21]. Overall, the signaling and content of ABA and GA play a critical role in regulating seed dormancy.

GA is required in seed development, and exogenous GA₃ has been applied to break seed dormancy. Exogenous GA₃ facilitates the germination of Fraxinus hupehensis seeds by enhancing the level of soluble sugars and weakening lipolysis [22]. Similarly, exogenous GA₃ might increase starch hydrolysis by stimulating the catalytic activity to mitigate oxidative damages in the early germination of Zanthoxylum dissitum seeds [23]. It has been found that exogenous GA₃ application prompts GA signal transduction and suppresses ABA synthesis to facilitate rice seed germination under low-temperature conditions [24]. Exogenous GA₃ might break seed dormancy and promote seed germination. However, little information is available about the response of dormancy release in recalcitrant seed to exogenous GA₃.

Panax notoginseng (Burkill) F. H. Chen (Sanqi in Chinese), a traditional Chinese medicinal plant, is a perennial herb from the family of Araliaceae[25]. Its seeds are classified into the group of morphophysiological dormancy (MPD), moreover it has been characterized by the typically recalcitrant trait that shows a high water content during the after-ripening process. The seeds need to undergo about 45 ~ 60 days of after-ripening process before germination (Duan et al., 2010). A preliminary study has demonstrated that incompletely developed embryos might result in the dormancy of P. notoginseng seed, and the embryo in the postharvest seed at a heart-shaped period has to be further differentiated and developed during the after-ripening process (An et al., 2006). Meanwhile, soluble sugar, starch and protein gradually decrease with the prolonged storage time in the seeds of P. notoginseng during the after-ripening process (An et al., 2010; Cui et al., 2010). PE2, GAI, KS, PP2C, GA2OX and other genes have been identified as the key genes involved in regulating the released dormancy of P. notoginseng seeds [26]. Our recent work has shown that exogenous GA₃ treatment could effectively shorten the after-ripening process and stimulate seed germination of P. notoginseng seeds (Ge et al., 2020). However, it is still unknown about the mechanisms through that GA₃ facilitates the germination of postharvest P. notoginseng seeds at the physiological and molecular levels.

In this study, we hypothesized that exogenous GA₃ application would enhance endogenous GA biosynthesis and accumulation, inhibit the expression of gene related to ABA signal transduction, and contribute to the embryo development, cell wall relaxation, and radicle protrusion, consequently shortening after-ripening process and promoting recalcitrant seed germination. Based on this hypothesis, we compared germination rate, hormonal content, and transcriptomic-related indicators of seeds treated with exogenous GA₃. We also identified differentially expressed genes (DEGs) associated with seed germination and the expression of genes related to seed germination verified by qRT-PCR. It would provide theoretical guidance for the application of recalcitrant seeds in agricultural production and seed storage.

Effects of exogenous GA ₃ treatment on embryo development and seed germination in P. notoginseng during the after-ripening process

Embryo development and seed germination in 50 mg L^− 1 GA₃-treated (LG), 250 mg L^− 1 GA₃-treated (MG), 500 mg L^− 1 GA₃-treated (HG) and control (CK) were as shown in Fig. 1. At 0 DAR, the embryos were enclosed by endosperms (Fig. 1A). The size of the embryos generally increased as the after-ripening process was prolonged (Fig. 1A-B). The embryo length was more than half of the seeds sections at 30 DAR, and the rates of Em/En of 50 mg L^− 1, 250 mg L^− 1and 500 mg L^− 1 GA₃-treated seeds were 61.98%, 60.50% and 63.01%, respectively. At 45 DAR, the 500 mg L^− 1 GA₃-treated the seeds had a significantly higher Em/En rate (92%) than 50 mg L^− 1 GA₃-treated ones and the control (Fig. 1B). Beginning 30 days after treatment, GA₃-treatment significantly enhanced the germination rate of P. notoginseng seeds (Fig. 1D). Compared with 30 DAR, the seed germination rate in control was raised by 10.0% and 27.0% at 45 DAR and 60 DAR, respectively, while the increase in the 50 mg L^− 1 GA₃-treated seeds were 30.0% and 53.0%, respectively, during the same period (Fig. 1D). Compared with CK, the external application of GA₃ significantly promoted seed germination of P. notoginseng, the rate of seed germination tended to raise as the GA₃ application increased (Fig. 1C).

RNA sequencing and gene annotation of the transcriptome

To acquire a general overview of the regulation of seed germination as impacted by exogenous GA₃ treatment with different concentrations, 27 cDNA libraries were selected to examine the transcriptome level of gene expression in P. notoginseng seeds. A total of 187.75 gigabytes (Gb) clean sequencing data was acquired (Table S1). For each sample, the average clear data was about 6.95 Gb, and Q30 (the percentage of bases with Phred > 30 to the total bases, Phrede = -10log₁₀(e)) was about 94%. The data were used for comparisons with the reference genome of P. notoginseng [27]. 86.09% ~ 88.82% of the reads in the 27 libraries were uniquely mapped by alignment with the reference genome of P. notoginseng (Table 1). The transcript abundances of genes were assessed by fragments per kilo base of exon per million fragments mapped (FPKM). The distribution of the log₂ (FPKM + 1) showed relatively high gene expression as shown in Fig. S2. In this experiment, all R² values between the three biological replicates were closer to 1 by using the Persons Correlation Coefficient (R) (Fig. S3), showing that the biological replicates of each sample had strong correlations.

Table 1

Comparative genome statistics of samples.
sample	total reads	total map	unique map	multi map	mapping rate (%)
CK-0-1	46815702	42910019	40899591(87.36%)	2010428(4.29%)	91.66%
CK_0_2	46919124	42880938	40775136(86.91%)	2105802(4.49%)	91.39%
CK_0_3	45916162	42104871	40017119(87.15%)	2087752(4.55%)	91.70%
LG_0_1	46769434	42497124	40923834(87.5%)	1573290(3.36%)	90.87%
LG_0_2	44727404	40601250	39100103(87.42%)	1501147(3.36%)	90.77%
LG_0_3	43111468	39537923	37614568(87.25%)	1923355(4.46%)	91.71%
HG_0_1	46684610	42938303	40777893(87.35%)	2160410(4.63%)	91.98%
HG_0_2	45715822	41919574	39764745(86.98%)	2154829(4.71%)	91.70%
HG_0_3	51812850	47721232	45354018(87.53%)	2367214(4.57%)	92.10%
CK_30_1	46770276	43229185	41469671(88.67%)	1759514(3.76%)	92.43%
CK_30_2	46087118	42530822	40909769(88.77%)	1621053(3.52%)	92.28%
CK_30_3	50775950	46563721	44146740(86.94%)	2416981(4.76%)	91.70%
LG_30_1	44439024	41026549	39462059(88.8%)	1564490(3.52%)	92.32%
LG_30_2	46805200	43226074	41570186(88.82%)	1655888(3.54%)	92.35%
LG_30_3	44743018	40935834	39281645(87.79%)	1654189(3.7%)	91.49%
HG_30_1	48011460	44131637	42367279(88.24%)	1764358(3.67%)	91.92%
HG_30_2	47257916	43528792	41720947(88.28%)	1807845(3.83%)	92.11%
HG_30_3	46156508	42433078	40956718(88.73%)	1476360(3.2%)	91.93%
CK_50_1	46450880	41971359	39989166(86.09%)	1982193(4.27%)	90.36%
CK_50_2	44037090	40404986	38490509(87.4%)	1914477(4.35%)	91.75%
CK_50_3	49358510	45429117	43281381(87.69%)	2147736(4.35%)	92.04%
HG_50_1	44787148	40771025	39010023(87.1%)	1761002(3.93%)	91.03%
HG_50_2	48294500	44388897	42618190(88.25%)	1770707(3.67%)	91.91%
HG_50_3	42958006	39350379	37781609(87.95%)	1568770(3.65%)	91.60%
LG_50_1	45946054	42287132	40654703(88.48%)	1632429(3.55%)	92.04%
LG_50_2	45088374	41649863	40047536(88.82%)	1602327(3.55%)	92.37%
LG_50_3	45204254	41657387	39812720(88.07%)	1844667(4.08%)	92.15%

Transcriptomic changes by treatment with exogenous GA₃

A total of 2971 and 9827 DEGs were common in P. notoginseng seeds with exogenous LG and HG treatment, respectively (Fig. 2B-C). Through pairwise comparisons, a total of 1064, 397, 1115, 2777, 792 and 6653 DEGs were identified at CK_0 d vs LG_0 d, CK_0 d vs HG_0 d, CK_30 d vs LG_30 d, CK_30 d vs HG_30 d, CK_50 d vs LG_50 d and CK_50 d vs HG_50 d, respectively (Fig. 2A-B). For DEGs in LG-treated seeds at 0 DAR, 497 were up-regulated and 567 down-regulated (Fig. 2B). In HG-treated seeds at 0 DAR, 265 were up-regulated and 132 down-regulated. At 30 DAR, 465 genes were up-regulated and 650 genes were down-regulated on the CK compared with LG-treated seeds; 1144 genes were up-regulated and 1633 genes were down-regulated on the CK compared with HG-treated seeds (Fig. 2A). At 50 DAR, 474 genes were up-regulated and 318 genes were down-regulated on the CK compared with LG-treated seeds (Fig. 2A); 3187 genes were up-regulated and 3466 genes were down-regulated on the CK compared with HG-treated seeds. To obtain the functional annotations of DEGs, GO annotation analysis was performed on DEGs (Fig. 3). The results showed that the most of DEGs were enriched in biological process and molecular function, while a number of DEGs were enriched in the cellular components in the comparisons at CK_30 d vs LG_30 d, CK_30 d vs HG_30 d, CK_50 d vs LG_50 d and CK_50 d vs HG_50 d (Fig. 3, Fig. S4). In biological process classification, these DEGs were specifically involved the metabolic processes, cells biological processes, and response the stress and abiotic stimulus. The molecular functions mainly included binding, catalysis, and transport activity. Besides, most of the gene products were located in cells and organelles.

Moreover, KEGG analysis was used to evaluate the biological functions of the DEGs (Fig. 4). In the comparisons at CK_0 d vs LG_0 d and CK_0 d vs HG_0 d, the DEGs were mainly identified in pentose and glucuronate interconversions, cyanoamino acid metabolism and protein processing in the endoplasmic reticulum (Fig. S5). In the comparisons at CK_30 d vs LG_30 d and CK_30 d vs HG_30 d, our results found that DEGs were mainly identified in plant hormone signal transduction, galactose metabolism, and amino sugar and nucleotide sugar metabolism. Importantly, in the comparisons at CK_50 d vs LG_50 d and CK_50 d vs HG_50 d, the results showed that the top KEGG pathways enriched were the plant hormone signal transduction, carbon metabolism and citrate cycle (Fig. 4C). Thus, DEGs functional enrichment suggested that plant hormone signal transduction and carbon metabolism were closely involved in GA₃-promoted seed development, and these pathways were further investigated.

Changes in profiles of plant hormone signal transduction and related metabolites in response to exogenous GA ₃ treatment

In Fig. 5, the map showed the expression pattern of DEGs related to plant hormone ABA and GA signal transduction. Compared with the CK, GA₃-treatment up-regulated DEGs related to GA biosynthesis and signal transduction during the after-ripening process, including ent-copalyl diphosphate synthase (CPS), GA20-oxidase (GA20ox) and GA INSENSITIVE DWARF1 (GID1), whereas down-regulated DELLA (Fig. 5A-B). Further, as shown in Fig. 5D, GA₃-treatment down-regulated most of the DEGs involved in ABA transport signal transduction pathway among them are Pyrabactin resistance 1-like (PYL) and ABSCISIC ACIDINSENSITIVE 5 (ABI5), whereas upregulated Protein Phosphatase 2C (PP2C). To explore the functions of endogenous hormones in seed germination, ABA, GA₃ and IAA contents in the CK and GA₃-treated P. notoginseng seeds during the after-ripening process were detected using LC-MS (Table 2). Compared with the CK, endogenous hormones GA₃ content was significantly the highest at 0 DAR, but IAA content was not changed considerably in the GA₃-treated group. By contrast, ABA contents were the lowest in the GA₃-treated group. Thus, GA₃ treatment could stimulate seed germination by altering the accumulation of endogenous hormones. Moreover, exogenous GA₃ induced seed germination through increased GA₃ concentration and decreased ABA concentration. These results imply that exogenous GA₃ enhances GA biosynthesis and accumulation, while inhibits the gene expression related to ABA signal transduction.

Table 2

Changes of endogenous hormones in the control and GA₃-treated *P. notoginseng* seeds during after-ripening process.
Endogenous hormone (ng/g)	Time (d)	Sample
Endogenous hormone (ng/g)	Time (d)	CK	GA₃ 50mg/L	GA₃ 500mg/L
GA₃	0	0.432 ± 0.078	4.006 ± 0.358	27.851 ± 2.118*
	30	0.197 ± 0.059	2.914 ± 2.582	15.118 ± 2.277*
	50	0.293 ± 0.244	0.788 ± 0.073	9.100 ± 0.948*
ABA	0	0.250 ± 0.045	0.228 ± 0.044	1.050 ± 0.474
	30	0.154 ± 0.013	0.154 ± 0.023	0.182 ± 0.016
	50	0.116 ± 0.026	0.105 ± 0.007	0.102 ± 0.007
IAA	0	20.465 ± 1.548	19.608 ± 1.091	19.756 ± 1.652
	30	12.695 ± 1.106	13.513 ± 2.230	11.191 ± 0.930
	50	7.108 ± 2.390	7.812 ± 1.471	8.167 ± 1.464
The values are means ± SD(n = 3). *P < 0.05, significant differences between the control and GA₃ treatments.

The expression of genes related to embryo development and cell wall relaxation in response to GA ₃ treatment

The expression of gene involved in embryo development and cell wall loosening in P. notogensing seeds during the after-ripening process showed significant changes under exogenous GA₃ treatment. As shown in Fig. 6A, we focused on embryo development-related and cell wall-related DEGs, which were clustered into five gene groups: Late Embryogenesis Abundant (LEA), Leafy Contyledon1(LEC1), Expansin (EXP), Xyloglucan Endotransglucosylase (XET) and Pectin Methylesterase (PME). Compared with the CK, the expression level of PME and LEA did not change upon GA₃ treatment at 0 DAR, while GA₃ increased the expression level of LEC1 and EXP. With the prolonged after-ripening process, LEA and PME were dramatically up-regulated in response to GA₃ applications. The expression of LEC1, EXP and other genes were found to be increased during after-ripening process after GA₃ treatment.

Verification of expression of DEGs using qRT-PCR

To test the reliability and the repeatability of RNA-seq, the DEGs related to GA biosynthesis and signal (CPS, GA20ox, DELLA), ABA signal and response (PYL, ABI5), embryo development (LEA) and cell wall metabolism (PME) were chosen for the confirmation of gene expression (Fig. 7). It indicated that the results of transcriptomic were reliable and accurate.

Exogenous GA ₃ effectively shortens the after-ripening process and promotes seed germination of P. notoginseng

Seed dormancy and germination are influenced by plant hormonals and the external environment [28–30]. Exogenous GA₃ treatment could significantly promote germination in wild species of pistachioo [8, 31]. It has been found that seed germination is substantially promoted by exogenous GA₃ treatment in Fraxinus hupehensis [22]. Consistently with the finding of previous studies, the present study revealed that exogenous GA₃ obviously promote seed germination of P. notoginseng (Fig. 1C-D). Compared with CK, the seed germination rate tended to be raised as GA₃ application increased, and the germination rate was highest in P. notoginseng seeds treated with 500 mg L^− 1 exogenous GA₃ (Fig. 1D). It has also been recorded that Acer mono Maxim. seeds are treated with 200 mg L^− 1 GA₃ and their germination rate effectively increased [32]. Nitraria tangutorum Bobr. seeds are treated with 150 mg L^− 1 GA₃ and germination rate, germination index and vigor index are the highest (Guo and Lin, 2009). These studies indicate that the appropriate concentration of exogenous GA₃ is one of the key factors in breaking dormancy and promoting seed germination. In our research, we verified the impacts of different concentrations of GA₃ (LG, MG and HG) on seed embryo development, germination rate and after-ripening process of P. notoginseng seeds (Fig. 1B-D), and found that the germination rate of P. notoginseng seed is gradually elevated with the increase of exogenous GA concentration (Fig. 1), indicating that 500mg L^− 1 of exogenous GA₃ is most appropriate to promote seed germination of recalcitrant P. notoginseng seeds.

GA essentially stimulates endosperm weakening and embryo expansion [33], and promotes the protrusion of radicle by breaking through the confines of the seed coat [34, 35]. Herein, we found that the endosperm tissue around seed embryos treated with HG is softened at 30 DAR compared with CK (Fig. 1A), implying that GA₃ treatment might stimulate the softening of tissues around seed embryo, thus providing sufficient space for embryo development. Our results agreed with the observation that GA accelerates the growth potential of the embryo and weakens the structures surrounding the embryo in tomato [36]. Above all, our results reveal that 500 mg L^− 1 GA₃ treatment might effectively shorten the after-ripening process and promote seed germination by stimulating seed embryo development and softening the tissues around the embryos of P. notoginseng.

Exogenous GA ₃ application accelerates P. notoginseng seed germination by changing endogenous hormone accumulation

ABA and GA antagonistically regulate seed dormancy and germination [28, 37]. The induction and maintenance of dormancy are positively regulated by ABA, while germination is enhanced by GA [38]. Consistently, our results showed that GA₃ content is highest, and ABA content is the lowest in P. notoginseng seeds treated with GA₃ (Table 2). This is agreement with other study showing an increase in energy requirements and endogenous GA₃ content but a decrease in ABA content during germination and growth of seeds[22]. Compared with the CK, our study found that the GA₃ content in P. notoginseng seeds had a 60-fold increase after treatment with 500 mg L^− 1 GA₃, followed by a 9-fold increase in seeds after treatment with 250 mg L^− 1 GA₃ at 0 DAR (Table 2). The levels were much too high to be endogenous GA₃ and they were reduced with time after treatment. It could be the result that the penetration of exogenous GA₃ into the seed tissues was caused by the concentration difference between the soak solution and the cytolymph during the soak treatment. Those results indicated that a part of measured endogenous GA₃ is likely to be remaining from the GA₃ treatment, but both of them contribute to alter the ratio of GA and ABA. Besides, our results found that exogenous GA₃ application could not cause auxin (IAA) content to be different in P. notogensing seeds, and this is contrary to the finding that exogenous GA₃ increases IAA content in the tiller node of rice (Oryza sativa L.) [39], implying that IAA responds diversely in the regulation network of plant development upon GA₃ treatment. Thus, we consider that exogenous GA₃ release dormancy to promote seed germination mainly through changing the ratio of GA and ABA.

Cellular ABA and GA levels are controlled by the balance between their biosynthesis and catabolism [40]. Our transcriptomic analysis revealed that the total of 2971 and 9827 DEGs are dramatically affected by exogenous LG and HG treatment, respectively (Fig. 2A). Meanwhile, it was significantly enriched for plant hormone signal transduction and related metabolic pathways regulated by GA (Fig. 4C-D), suggesting that GA induces dramatic responses at the transcriptional level. Some candidate genes in GA₃ and ABA signaling pathways also determine seed germination [37, 41]. In our study, the expression level of CPS, GID1 and most of GA20ox were downregulated by GA₃ treatment at 0 DAR (Fig. 7C-D), and this effect gradually weakens and was lost with decreasing levels of GA₃ in seeds (Table 2), suggesting that high concentrations of GA₃ in treated seeds might be a negative regulator to suppress GA biosynthesis and signaling by reducing expression of some GA-biosynthesis genes in a homeostasis mechanism (Binenbaum et al., 2018). A study on barley, wheat and rice has shown that HvGA20ox is a pivotal gene for regulate seed germination in barley [42]. OsGA20ox2 and OsGA2ox3 were essential genes to control seed germination in rice [43], and the mutation OsGA20ox2 shows the reduced GA level and enhanced seed dormancy [44]. Likewise, our study found that CPS, GA20ox, GID1 and DELLA genes involved in GA hormone biosynthesis and catabolism pathways are affected by exogenous GA₃ treatment (Fig. 5A-B). GA₃ upregulated the expression of CPS, GA20ox and GID1, and downregulated DELLA at 30 DAR and 50 DAR (Fig. 7C-D). DELLA is a plant growth suppressor, while GID1 is a receptor for GA₃, it acts by binding to GID1 receptor to degrade DELLA protein in plants [45, 46]. Overall, the expression levels of GA20ox and GID1 were upregulated, and the expression level of DELLA was downregulated by GA₃ treatment during the after-ripening process, thereby perturbing GA₃ signal transduction in recalcitrant P. notogensing seeds.

A comparative analysis of PP2C mutants suggests that AtPP2CA is a significant player in seeds [47, 48]. Of these, the ABA receptors PYR1/PYL proteins might confer a prominent function in seed ABA responsiveness through regulating PP2C activity [49, 50], and the pyr1 prl1 prl2 prl4 quadruple mutant shows ABA insensitive the germination [49]. Genetic analysis reveals that ABA-INSENSITIVE 3(ABI3), ABA-INSENSITIVE 4 (ABI4) and ABA-INSENSITIVE 5 (ABI5) are the key transcription factors that confer seed ABA responsiveness [51]. The seeds of abi5 mutants reduce transcript levels of Early Methionine-labelled 1 and 6 (EM1 and EM6), which are associated with germination process [52, 53]. The transcriptomic analysis showed that the expression of PYL, PP2C and ABI5 has a significant change in P. notoginseng seeds treated with exogenous GA₃ (Fig. 5C). PYL and ABI5 showed a higher expression level at the 0 DAR, and they gradually decreased with the prolonged after-ripening process in P. notoginseng seeds. Surprisingly, compared with CK, the expression of PYL and ABI5 tended to decline as the GA₃ application increased, and it was lowest in P. notoginseng seeds treated with 500 mg L^− 1 exogenous GA₃ at 50 DAR (Fig. 7E-F), and the expression trend of PP2C was reversed during the after-ripening process (Fig. 5C). Our results suggest that exogenous GA₃ regulates the essential genes to perturb endogenous GA and ABA biosynthesis and catabolism in P. notoginseng seeds. This might partly contribute to the antagonistic action of GA and ABA on seed germination and growth.

The elevated endogenous hormone GA effectively promote the expression of genes related to embryo development and cell wall loosening

The synthesis and catabolism of GA₃ vigorously promotes cell division during seed development and germination [54, 55]. A previous study has shown that the incomplete development of embryos could result in seed dormancy of P. notoginseng [56]. Recent studies revealed that LEAFY COTYLEDON 1 (LEC1) is a critical regulator of seed development, its loss of function results in a short embryo axis and intolerance to desiccation [57, 58]. Consistently, Late Embryogenesis Abundant (LEA) and LEC1 are required for seed maturation and acquisition of desiccation tolerance [59, 60]. In our study, we found that the expression of LEC1 and LEA is lower in CK at 0 DAR, and they are dramatically up-regulated in response of P. notoginseng seeds to GA₃ applications during the after-ripening process (Fig. 6A), demonstrating that the embryo development is relatively vigorous under GA₃ treatment (Fig. 1A). These results support the view that GA₃ treatment could promote the embryo development to boost seed germination of postharvest P. notogensing.

The architecture of cell wall is a key determinant for plant growth [61]. The dormancy or germination is determined by the balance between the resistance strength of the surrounding tissues and the growth potential of the elongating radicle [62]. There are a series of evidence that GA₃ could facilitate radicle protrusion by breaking through the mechanical constraints of the seed coat during seed germination [54, 63]. The cell wall-degrading enzymes, such as cellulases, xyloglucan endotransglucosylase-hydrolase (XTH), pectinesterase (PME), expansins (EXP) and hemicellulases, have been proved to contribute to cell wall loosening [61, 64–66]. In our study, DEGs (PME, EXP and XTH) involved in cell wall development were up-regulated by exogenous GA₃ treatment (Fig. 6A-B). Our result is consistent with observations that xyloglucan endotransglucosylase (XET), xyloglucan endohydrolase (XEH) and EXP are upregulated during Arabidopsis seed germination [67, 68]. Our observations confirmed that a series of cell wall-degrading genes is up-regulated significantly in P. notoginseng seeds treated with exogenous GA₃ (Fig. 7G), and it suggests that exogenous GA₃ might promote cell wall metabolism and endosperm degradation. Compared with CK, the expression of EXP and PME were both upregulated in P. notoginseng seeds treated with LG and HG. Differently, the up-regulated expression level in the seeds treated with HG was significantly higher than those treated with LG (Fig. 6B). Thus, although LG, MG and HG treatments all promote seed germination, to a higher degree HG accelerate the degradation of cell wall to create more spaces for seed germination by up-regulating EXP and PME. This could be regarded as the reason for the highest germination rate of P. notoginseng seeds treated with HG. In general, we believe that the genes (LEA, LEC1, EXP, PME and XEH) involved in embryo development and the cell wall degradation might create more spaces for radicle elongation to accelerate the germination in postharvest P. notoginseng seeds treated with GA₃.

Exogenous GA₃ increases the content of endogenous hormones GA₃ through permeation and alters the ratio of GA and ABA to promote seed germination of P. notogensing. GA-treated P. notogensing seeds maintain higher development and germination than CK treatment. We also find that GA₃ upregulate DEGs involved in GA biosynthesis and catabolism, embryo development and the cell wall loosening, while downregulate ABA biosynthesis and catabolism. Based on the findings of the present work, a model is proposed to explain the dormancy mechanism in recalcitrant P. notogensing seeds regulated by GA₃ (Fig. 8). Exogenous GA₃ application increases the content of endogenous hormones GA₃ through permeation to alter the ratio of GA/ABA, and this would contribute to the development of the embryo, break the mechanical constraints of the seed coat and promote the protrusion of the radicle in postharvest recalcitrant seeds. These findings would comprehensively improve our understanding of the potential roles of GA in regulating the dormancy of recalcitrant seeds during the after-ripening process.

Materials and treatments

Seeds were routinely harvested from the plants of 3-year-old P. notoginseng (Fig. S1A) that were cultivated at the experimental farm of Wenshan Miao Xiang P. notoginseng Industrial Co., Ltd., China (Longitude 104°32′, latitude 23°53′). In November, 3-year-old mature and plump seeds of P. notoginseng were selected (Fig. S1B). After artificial peeling (Fig. S1C), the seeds were washed in ddH₂O, soaked and disinfected with 5% CuSO₄ bactericidal solution and washed twice, then the seeds were obtained by indoor shade drying out surface moisture. The seeds were submitted to one of three different treatments by soaking for 24 h, the concentrations 50 mg L^− 1 (LG), 250 mg L^− 1 (MG) and 500 mg L^− 1 (HG) of exogenous hormones GA₃ were selected for soaking treatment, and ddH₂O-soaked treatment was defined as the control (CK). The soaked seeds with three replicates were placed in a ventilated net basket for 50 days in a sandy stratification chamber at 15 ± 5℃ to accomplish after-ripening process. The start point before the seeds was stored in sandy stratification is defined as the time point of 0 days after-ripening (DAR). At each sampling point (0, 30, 50 DAR), the samples from the concentrations of 50 mg L^− 1 (LG) and 500 mg L^− 1 (HG) of GA₃ were selected based on the results from experiments (Fig. 1) for further assays.

Microscopic inspection and morphological measurements

A number of seeds were fixed using FAA (70% alcohol: acetic acid: formalin = 18:1:1) for microscopic inspection. The determination of embryo (Em), endosperm (En) and embryo rate (Em/En) was carried out at four time points of 0, 15, 30 and 45 DAR. Seeds were divided in half lengthwise by using a razor blade. Seed sections were examined using a stereoscopic microscope (ZEISS, SteREO Discovery.V20, Germany) equipped with a digital camera. The pictures were processed (brightness and contrast adjusted) and combined using Photoshop CS6 (Adobe, USA).

HPLC-MS analysis of endogenous hormone GA₃, ABA and IAA content

Samples used for the determination of endogenous hormone content were frozen in liquid nitrogen at three time points of 0, 30 and 50 DAR. Endogenous hormone ABA, GA₃ and IAA content in seed of P. notoginseng was examined as reported by Pan et al [69] with some modification.

Total RNA extraction and transcriptome analysis

For LG, HG and CK treatment, the sample at 0, 30 and 50 DAR were separated and applied for RNA extraction. Using the Plant Plus Kit (Tiangen, Beijing, China) to extract the total RNA with three replications, and RNA quality was monitored on 1% agarose electrophoresis. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), and RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Using NEBNext® UltraTM RNA Library Prep Kit, sequencing libraries were generated for Illumina® (NEB, USA) following the manufacturer’s recommendations and index codes were added to attribute sequences. The libraries were sequenced on an Illumina platform by the Novegene Technology Company (Beijing, China). Raw reads were analyzed and low quality reads and reads containing adapters were removed, resulting in clean reads. The clean reads were mapped to the P. notogensing reference genome (Fan et al., 2020) using HISAT2 v2.0.5, and novel genes prediction were made with String Tie (1.3.3b) [70].

Differential expression analysis of paired conditions with three biological replicates per condition was performed using the DESeq2 R package (1.16.1). According to the method of Benjamini and Hochberg [71], the P-values were adapted to control the false discovery rate. Genes with an adjusted P-value < 0.05 found by DESeq2 were designated as differential expression. Gene Ontology (GO) analysis and KEGG analysis of differentially expressed genes (DEGs) were performed by the clusterProfiler R package. The terms with corrected P-value < 0.05 were identified as significant enrichment of differently expressed genes.

Gene expression assessment

Gene expression was assayed by Quantitative Real-time PCR (qRT-PCR). The total RNA was isolated from seeds of P. notogensing samples using the same scheme described for RNA-Seq. The cDNA was synthesized by using Prime Script RT reagent kit (Takara Bio, Kyoto, Japan). As shown in Table S2, primers were accessed using Premier 3.0 [72] for quantitative PCR (qRT-PCR) and synthesized by Tsingke Biotech Co., Ltd. (Kunming, China). The qRT-PCR reaction was performed with three technical replicates using the Quant studio12 K Flex System (Thermo Fisher Scientific). The GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE(GAPDH) was chosen as the internal reference for P. notoginseng seeds. Candidate genes were analyzed for relative expression levels using the 2^−ΔΔCt algorithm[73] by standardizing their transcript levels of related genes in control. Each sample was analyzed in three replicates.

Statistical analysis

The experiment was performed in three biological replicates. Statistical analyses were carried out using the SPSS software package (Chicago, IL, USA) and SigmaPlot 10.0, where the variables were present as the mean ± SD (n = 3). The least significant difference (LSD) was calculated and P < 0.05 was deemed statistically significant.

ABA: Abscisic acid; ABI3: ABA-INSENSITIVE 3; ABI4: ABA-INSENSITIVE 4; ABI5: ABA-INSENSITIVE5; CPS: Ent-copalyl diphosphate synthase; CK: Control; DAR: Days after-ripening; DEGs: Differentially expressed genes; EXP: Expansin; Em: Embryo; En: Endosperm; Em/En: Embryo rate; GA: Gibberellin; GA₃: Gibberellic acid; GA20ox: GA 20-oxidase; IAA: Auxin; GO: Gene Ontology; LEA: Late Embyogenesis Abundant; LEC1: Leafy Cntyledon1; MD: Morphologcal; NCED: Nine-cis-epoxycarotenoid dioxygenase; PYL: Pyrabactin resistance 1-like; PME: Pectinesterase; PD: Physiological; PY: Physical; PY + PD: Combinational; PME: Pectin Methylesterase; PP2C: Protein Phosphatase 2C; XET: Xyloglucan Endotransglucosylase; XEH: Xyloglucan endohydrolase.

Ethics approval and consent to participate

Not applicable. The authors declared that a permission to collect Panax notoginseng material has been obtained, and experimental research works on the plants described in this paper comply with institutional, national and international guidelines.

Consent for publication

Not applicable.

Availability of data and materials

The raw sequencing data from this study have been deposited in the Genome Sequence Archive in BIG Data Center (https://bigd.big.ac.cn/), Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under the accession number: CRA008378. Other data generated or analyzed during this study are included in this published article and its supplementary information files. Hoo & Tseng firstly undertook the formal identification of the plant material Panax notoginseng (Burkill) (Journal of Systematics and Evolution 11: 435, 1973) in Flora of China.

Competing Interests

The authors report no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work is funded by the Major Special Science and Technology Project of Yunnan Province (202102AA310048); and the National Key Research and Development Plan of China (2021YFD1601003).

Authors’ contributions

Chen Jun-Wen directed the experiment and made suggestions for the writing of the manuscript. Ge Na wrote the manuscript, Yang Ling and Ge Na participated most of the experiments. Huang Rong-Mei, Jia Jin-Shang, Wang Qing-Yan, Chen Cui, Meng Zhen-Gui and Li Long-Geng analyzed the relevant experimental data. All authors contributed to the article and approved the submitted version.

Acknowledgements

We thank Dr JYZ for technical assistance. We are also grateful to KLL and MJM for their assistance with data analysis.

Authors' information

¹ College of Agronomy & Biotechnology, Yunnan Agricultural University, Kunming, Yunnan 650201, China. ²The Key Laboratory of Medicinal Plant Biology of Yunnan Province, Yunnan Agricultural University, Kunming, Yunnan 650201, China. ³ National & Local Joint Engineering Research Center on Germplasm Innovation & Utilization of Chinese Medicinal Materials in Southwestern China, Yunnan Agricultural University, Kunming, Yunnan 650201, China.

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