Abstract

   Panax japonicus, an endangered species in China, is usually used as a
   traditional medicine with functions of hemostasis, pain relief, and
   detoxify. However, the seeds of P. japonicus are hard to germinate in
   natural conditions, and the molecular events and systematic changes
   occurring in seed germination are still largely unknown. In this study,
   we compared the seeds in different germination stages in terms of
   morphological features, antioxidant enzyme activities, and
   transcriptomics. The results indicated that sand storage at 25℃ for 120
   d effectively released the seed dormancy of P. japonicus and promoted
   the seed germination. Moreover, sand storage treatment increased the
   antioxidant capacity of P. japonicus seeds through increasing the
   activities of SOD, POD, and CAT. The RNA-seq identified 28,908
   differentially expressed genes (DEGs) between different germination
   stages, of which 1697 DEGs significantly changed throughout the whole
   germination process. Functional annotations showed that the seed
   germination of P. japonicus was mainly regulated by the DEGs related to
   pathways of ROS-scavenging metabolism, plant hormonal signal
   transduction, starch and sucrose metabolism, energy supply (glycolysis,
   pyruvate metabolism, and oxidative phosphorylation), and
   phenylpropanoid biosynthesis, as well as the transcription factors such
   as bHLHs, MYBs, WRKYs, and bZIPs. This study provides a foundation for
   unveiling molecular mechanisms underlying the seed germination and is
   beneficial for accelerating the development of P. japonicus industry.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12870-024-05904-w.

Introduction

   Panax japonicus C.A. Meyer (P. japonicus) is an endangered species
   which mainly grows in China, Japan, and Korea. The rhizome of P.
   japonicus has been widely used as a traditional medicine [[42]1]. Due
   to the unique pharmacological activities of eliminating phlegm,
   anti-inflammatory, and antioxidant, P. japonicus has been included in
   the Pharmacopeia of the People’s Republic of China [[43]2]. According
   to our field investigation, excessive collection of wild resources and
   the seed dormancy induced low germination rate (≤ 10%) have resulted in
   low regeneration of P. japonicus in the wild, thereby limiting the
   rapid development of P. japonicus industry. Previous studies indicated
   that the embryo of fresh P. japonicus seed was just a cell mass
   composed of multiple cells, and it took 60–70 days to complete “seed
   embryo post-maturation” [[44]3]. Zhang et al. [[45]4] reported that the
   germination percentage of P. japonicus seed was 60% after wet sand
   storage at 5 ℃ with 70% relative humidity. In our previous research, we
   found that wet sand storage at room temperature (25 ℃) is an effective
   means to release the dormancy of P. japonicus seed [[46]5]. However,
   the morphological and physiological changes of P. japonicus are still
   unclear during the seed dormancy release and germination.

   Seed germination is regulated by a series of orderly physiological,
   biochemical, and molecular processes, including hormonal signal
   transduction, nutrient consumption, energy generation, and
   transcription activation [[47]6–[48]7]. Increasing information from
   transcriptomics studies has enhanced global understanding of the seed
   germination process and regulation. RNA sequencing (RNA-seq) is an
   effective tool to understand the molecular mechanisms of plant seed
   germination, and this technique has been used in exploring the seed
   germination of several medicinal plants like Cinnamomum migao,
   Cyclobalnopsis gilva and Fraxinus hupehensis [[49]8–[50]10].
   Nevertheless, the biological events occurred at transcriptional level
   in the seed germination of P. japonicus are still elusive. Few
   researches were conducted to reveal the dynamic transcriptome profiles
   of P. japonicus seed germination. Thus, the molecular mechanism
   underlying the seed germination of P. japonicus still remains unknown,
   which is not conducive to its sexual propagation.

   In this study, we conducted an indoor experiment to investigate the
   morphological, physiological, biochemical, and transcriptional changes
   of P. japonicus seeds during germination. For the first time, we
   performed transcriptome analysis to reveal the biological events
   occurred at different germination stages, which can help better
   understand the molecular mechanism underlying P. japonicus seed
   germination. In addition, this study benefits the propagation of P.
   japonicus and provides a foundation for further investigating the
   regulatory networks involved in the seed germination P. japonicus.

Materials and methods

Experimental materials and design

   Fresh mature fruits of P. japonicus were harvested from Xintang
   Township, Enshi, Hubei Province, China (109°46′41″ E, 30°11′57″ N,
   altitude 1600 m), in September 2021. After the skin and flesh were
   removed, seeds of P. japonicus were surface-sterilized with 3% NaClO
   for 30 min and rinsed 6–7 times with sterile distilled water (1 min
   each time) to remove visible floating particles and then stored in dry
   ventilated condition. After one week, the seeds were soaked in sterile
   distilled water until imbibing water to full imbibition (generally
   48 h). Then, the seeds were placed in wet sand (65% relative humidity)
   in a growth chamber at 25 ℃ with 12 h day-light (20 µmol m^− 2 s^− 1).
   Three periods of seed germination were investigated, namely, stage I
   (no sand storage, DS), stage II (sand storage for 60 days, RS), and
   stage III (sand storage for 120 days, GS) (Fig. [51]1). The number of
   germinated seeds was counted in different stages, with the radicle
   length exceeding 2 mm as the germination criteria. Each stage had three
   biological replicates with 500 seeds per replicate, and 100 seeds from
   each replicate were randomly collected for subsequent analysis. The
   samples (seed coat removed) were carefully washed with distilled water
   and dried with absorbent paper, then flash-frozen in liquid nitrogen
   and stored at -80℃ for measurement of physiological indexes and
   transcriptome analysis.

Fig. 1.

   [52]Fig. 1
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   Morphological characteristics of P. japonicus seeds. DS: dormant seeds;
   RS: ringent seeds; GS: germinated seeds

Morphological and physiological indexes

   The longitudinal sections of P. japonicus seeds were observed by a
   stereomicroscope (OLYMPUS SZ61) to learn the morphological changes of
   seed embryos during different germination stages. The length and width
   of seeds were measured with a vernier caliper, and the seed weight was
   determined with a precision balance (0.0001 g) [[54]11].

   The activities of antioxidant enzymes such as superoxide dismutase
   (SOD), peroxidase (POD), and catalase (CAT), were measured according to
   the previous studies [[55]12–[56]13]. The content of malondialdehyde
   (MDA) and superoxide anion (O[2]^−) was determined following the method
   described by Wang et al. [[57]14] and Zhang et al. [[58]15],
   respectively.

Library construction and transcriptome sequencing

   The nine cDNA libraries of P. japonicus seeds from different
   germination stages were constructed according to our previous study
   [[59]16]. All the libraries were sequenced by Shanghai Personal
   Biotechnology Co., Ltd. (Shanghai, China) based on Illumina Novaseq
   6000.

De novo assembly and functional annotation

   The de novo assembly and functional annotation were conducted according
   to our previous research [[60]16], and the GO and KEGG Enrichment
   analysis were performed to obtain a detailed description of the DEGs.

Analysis of differentially expressed genes

   In this study, differentially expressed genes (DEGs) were screened
   according to the previous studies [[61]9, [62]16]. The hierarchical
   clustering of the DEGs was performed with p < 0.05, which indicated
   that the cluster distribution was significant. The pathway map images
   presented in this study are obtained from KEGG [[63]17], and we have
   got the copyright permission to modify the KEGG images depicted in
   figures.

qRT-PCR analysis

   To verify the accuracy of the RNA-Seq results, ten DEGs with different
   expression patterns were verified via qRT-PCR. The 18 S rRNA gene was
   used as reference gene according to a previous report [[64]18]. The
   qRT-PCR reaction was performed according to the method described in our
   previous study [[65]16]. The relative expression of specific genes was
   calculated using the 2^−ΔΔCt method [[66]19]. All the primers used are
   listed in Table [67]S1.

Statistical analysis

   The means and deviations of the biological replicates (n = 3) were
   calculated using Microsoft Excel 2019. Statistics and correlation
   analysis were performed using SPSS 22.0 (Chicago, Illinois). The
   differences between the samples were determined by Duncan’s multiple
   range test at p < 0.05. The graphs were built using origin 2021 (Origin
   lab, Northampton, Ma, USA). Heatmaps and hierarchical cluster analysis
   were performed using TBtools (Version 2.001) [[68]20].

Results

Morphology and anatomical structure

   The seeds of P. japonicus had a mean length of 4.96 mm, width of
   3.63 mm, and thousand seed weight of 36.80 g. Average moisture content
   of seeds was 54.64% (Table [69]1). The morphological characteristics
   and anatomical structures of P. japonicus seeds were observed in
   different germination stages. The seed coat is light yellow, with a
   punctiform micropylar pore. The endosperm is white with loose
   structure. From the DS to GS, the seed embryo developed quickly and
   elongated obviously until filling almost the whole endosperm. Moreover,
   the colour of cotyledon changed from white to light yellow. With the
   increase of germination time, the radicle protruded the seed coat and
   reached 2 mm at the GS (Fig. [70]1).

Table 1.

   The basic information of P. japonicus seeds
   Item Mean Standard deviation Amplitude Coefficient of variation (%)
   Thousand seed weight (g) 36.80 0.16 0.29 4.35
   Moisture content (%) 54.64 0.38 0.71 0.70
   Length (mm) 4.96 0.55 2.42 11.08
   Width (mm) 3.63 0.40 1.62 11.02
   [71]Open in a new tab

The physiological and biochemical indexes and germination percentage of P.
japonicus seeds

   The results showed that wet sand storage effectively released the seed
   dormancy of P. japonicus, and the germination rate reached 89% in the
   GS (Fig. [72]2A). Compared with the DS, the activity of SOD
   (Fig. [73]2B) and the content of O[2]^− (Fig. [74]2C) were
   significantly increased in the GS, while the CAT (Fig. [75]2D) and POD
   (Fig. [76]2E) activities, as well as the MAD content (Fig. [77]2F) were
   significantly increased in both RS and GS (p < 0.05).

Fig. 2.

   [78]Fig. 2
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   The germination percentage (A), CAT, POD, and SOD activities (B-D), as
   well as MDA and O[2]^− levels (E-F) of P. japonicus seeds in different
   germination stages. DS: dormant seeds; RS: ringent seeds; GS:
   germinated seeds

RNA-Seq and de novo assembly

   Nine cDNA libraries were constructed from the P. japonicus seeds
   sampled in different germination stages and sequenced using the
   Illumina Novaseq 6000. High-quality reads were obtained after removing
   low-quality reads and adaptor sequences (Table [80]S2). A total of
   41.81, 42.66, and 38.59 million raw reads, as well as 39.17, 40.12, and
   38.37 million clean reads were obtained from DS, RS and GS groups,
   respectively. Accordingly, a total of 117,204 unigenes were assembled,
   with an average length of 914 bp and the maximum length of 15,904 bp
   (Table [81]S3). Moreover, the Q20 values were 98.06%, 98.08%, 98.07%
   and the Q30 values were 94.04%, 94.12%, 94.40% for DS, RS, and GS,
   respectively (Table [82]S2). Thus, the assembly quality of the
   transcriptome was satisfactory.

Functional annotation of unigenes

   The functional annotations based on different databases were performed
   to obtain comprehensive information of the assembled unigenes.
   Specifically, 50,518 (41.10%) annotated unigenes were obtained from NR
   database; 31,493 (26.87%) annotated unigenes were obtained from GO
   database; and 21,526 (18.37%), 25,725 (21.95%), 49,569 (42.29%), and
   37,194 (31.73%) were obtained from KEGG, Pfam, eggNOG, and Swissprot
   databases, respectively (Table [83]S4).

   GO is a gene function database. 31,493 unigenes were classified into
   three GO categories including “biological process (BP)”, “molecular
   function (MF)” and “cellular components (CC)” (Fig [84]S1, Table
   [85]S5). The cellular component category mainly comprised “cellular
   anatomical entity” (80.86%) and “protein-containing complex” (19.14%).
   In the molecular function category, “binding” (41.80%), “catalytic
   activity” (35.67%) and “transporter activity” (5.66%) were
   predominantly enriched. In the biological process category, “cellular
   process” (21.11%), “metabolic process” (20.17%) and “biological
   regulation” (11.14%) were highly represented.

Comparative analysis of DEGs

   Differential expression analysis of unigenes was performed to obtain
   the transcriptional dynamic expression pattern during the seed
   germination of P. japonicus (Fig. [86]3). Compared with the DS, the
   upregulated genes in the RS and GS were 12,105 and 8804, respectively,
   while the downregulated genes were 4617 and 6324, respectively (Table
   [87]S6). The diagram showed that 16,722, 15,128, and 17,706
   differentially expressed genes (DEGs) were obtained from “DS_vs_RS”,
   “DS_vs_GS”, and “RS_vs_GS” comparisons, respectively (Fig. [88]3A).
   Interestingly, a total of 1614 DEGs significantly changed throughout
   the whole germination process (Fig. [89]3B), thus indicating these DEGs
   may be responsible for P. japonicus seed germination. Furthermore, a
   total of 28,908 DEGs (union set of all DEGs) were compared between DS,
   RS, and GS (Fig. [90]3C). Moreover, the genes in different groups (DS,
   RS, and GS) showed differential expression profiles.

Fig. 3.

   [91]Fig. 3
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   Differentially expressed genes (DEGs) in P. japonicus seeds under
   different germination stages. A. Statistical analysis of
   up/downregulated genes in the DS, RS and GS. B. Venn diagram of all the
   DEGs. C. Hierarchical clustering of all the DEGs

KEGG pathway analysis of DEGs

   The KEGG annotations indicated that the DEGs identified in the
   comparisons of “DS_vs_RS”, “RS_vs_GS”, and “DS_vs_ GS” were enriched
   into 132, 132, and 127 pathways, respectively (Table [93]S7-[94]S9).
   4566, 5067, and 3925 DEGs were identified at a significant level of
   p-value < 0.05 in “DS_vs_RS”, “RS_vs_GS”, and “DS_vs_ GS” comparisons,
   respectively, and these genes were significantly enriched in 14, 22,
   and 23 pathways, respectively (Table [95]S7-[96]S9). The top 30
   enriched KEGG pathways were analyzed at a significant level of p < 0.05
   (Table [97]S7-[98]S9). In particular, the phenylpropanoid biosynthesis
   (ko00940), plant hormone signal transduction (ko04075), starch and
   sucrose metabolism (ko00500), sesquiterpenoid and triterpenoid
   biosynthesis (ko00909), and photosynthesis (ko00195), etc., were
   significantly enriched in at least one of these three comparisons (Fig
   [99]S2), indicating that these pathways might play important roles in
   the seed germination of P. japonicus.

Hormonal signal transduction

   In this study, we performed KEGG pathway enrichment analysis for DEGs
   involved in hormonal signal transduction process (ko04075). The results
   showed that various hormonal signaling pathways, such as auxin,
   brassinosteroid, ethylene, gibberellin, cytokinin, abscisic and
   salicylic acid, were activated in the seed germination of P. japonicus
   (Fig. [100]4). Compared with the DS, most of the DEGs involved in the
   pathway of auxin signal transduction, such as AUXI, TIR1, SAUR, and
   AUX/IAA, were significantly upregulated in the RS or GS. In
   brassinosteroid signal transduction pathway, the BSK, BIN2, BZR1/2, and
   CYCD3 genes were significantly upregulated in both RS and GS, compared
   with the DS. All the genes encoding GID1 and most of the genes encoding
   DELLA were significantly downregulated in gibberellin signal
   transduction pathway under RS and GS, compared with the DS. Moreover,
   CRE1, AHP, B-ARR and A-ARR genes associated with cytokinin signal
   transduction pathway were greatly stimulated in the RS and GS. In
   addition, the TGA and PR-1 genes involved in salicylic acid signal
   transduction pathway were significantly upregulated in the RS or GS,
   compared with the DS. Contrarily, almost all the genes involved in the
   pathways of ethylene and abscisic acid signal transductions, were
   remarkably downregulated in both RS and GS, compared with the DS
   (Fig. [101]4).

Fig. 4.

   [102]Fig. 4
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   Heat map of DEGs involved in plant hormone signal transduction
   pathways. The relative expression levels of DEGs were calculated using
   the Log[2]FC

Starch and sucrose metabolism

   During the germination process, 71 and 102 DEGs involved in starch and
   sucrose metabolism pathway (ko00500) were identified from “DS_vs_RS”
   and “DS_vs_GS” comparisons, respectively ([104]S7-[105]S9). Compared
   with the DS, the genes encoding α-amylase (AMY), 4-α-glucanotransferase
   (DPE), α-glucosidase (AGLU), Glycogen phosphorylase (PHS), and
   Beta-glucosidase (BGL) were all upregulated in the RS and GS
   (Fig. [106]5), which might result in the degradation of starch in P.
   japonicus seeds. However, most of the genes encoding sucrose synthase
   (SUS) were downregulated in both RS and GS (Fig. [107]5), compared with
   the DS, which might decrease the accumulation of sucrose. In a word,
   the degradation of starch and sucrose was stimulated in the seed
   germination of P. japonicus.

Fig. 5.

   [108]Fig. 5
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   The DEGs related to starch and sucrose metabolism pathways. The
   relative expression levels of DEGs were calculated using the Log[2]FC

Energy supply

   Compared with the DS, multiple genes related to glycolysis (ko00010)
   and pyruvate metabolism (ko00620) were upregulated in the RS or GS
   (Fig. [110]6). Most of the genes encoding fructose-bisphosphate
   aldolase (ALF), galactose mutarotase (GALM), glyceraldehyde-3-phosphate
   dehydrogenase (G3PC), pyruvate kinase (KPY), and enolase (ENO) were
   remarkably upregulated in the RS and GS, compared with the DS
   (Fig. [111]6A). In addition, the expressions of genes encoding pyruvate
   kinase (KPY) and malate dehydrogenase (MDH) in the RS or GS were
   remarkably higher than those in the DS stage. Especially, the
   expression levels of genes encoding pyruvate dehydrogenase (ODP) and
   malate synthase (MASY) in the RS were 15- to 1260-fold higher than
   those in the DS, but there was no significant difference between DS and
   GS (Fig. [112]6B). On the whole, these key genes related to glycolysis
   and pyruvate metabolism exhibited higher expression levels in the RS or
   GS than those in the DS. Taken together, we presumed that 25 °C wet
   sand storage could facilitate the seed germination of P. japonicus by
   accelerating the glycolysis and pyruvate metabolism in the RS and GS.

Fig. 6.

   [113]Fig. 6
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   The DEGs involved in energy supply. A. DEGs in the pathway of
   glycolysis. B. DEGs in the pathway of pyruvate metabolism. C. DEGs in
   the pathway of oxidative phosphorylation. The relative expression
   levels of DEGs were calculated using the Log[2]FC

   In this study, 105 and 104 DEGs in the “DS_vs_RS” and “DS_vs_GS”
   comparisons ([115]S7-[116]S9), respectively, were found to be involved
   in oxidative phosphorylation (ko00190). Compared with the DS, the genes
   encoding NADH dehydrogenase (NDUS2, NDS5A, NDUS1, NDUA2, NDUB9, etc.),
   succinate dehydrogenase (SDHB, SDH3, and SDHA), cytochrome c reductase
   (QCR, QCR2, QCR6, CY1, UCR1, and CYB), cytochrome c oxidase (COX),
   pyrophosphate phospho-hydrolase (IPYR), and ATP synthase (ATPO, VHAA3,
   ATPF2, ATP9, ATP6, etc.) were significantly upregulated in the RS
   (p < 0.05) (Fig. [117]6C). These results showed that wet sand storage
   could accelerate the oxidative phosphorylation metabolism in the RS to
   release enough energy for the seed germination of P. japonicus.

Phenylpropanoid biosynthesis

   Besides the pathways of plant hormone signal transduction, starch and
   sucrose metabolism, and energy supply, the phenylpropanoid biosynthesis
   pathway (ko00940) also showed a significant change during the seed
   germination. 75 and 97 DEGs in the “DS_vs_RS” and “DS_vs_GS”
   comparisons, respectively, were found to be involved in phenylpropanoid
   biosynthesis pathway ([118]S7-[119]S9). Numerous genes encoding
   phenylalanine ammonia-lyase (PAL), cytochrome (C7A), dehydrogenase
   (ADH), methyltransferase (COMT), 3-O-(6-caffeoylglucoside) (5MAT), and
   peroxidase (POD) were significantly upregulated in the RS and GS
   (Fig. [120]7), while the genes encoding 4-coumarate–CoA ligase (4CL)
   were significantly downregulated in the RS and GS, compared with the
   DS. Thus, we speculated that the regulation of these genes mentioned
   above might play a pivotal role in the seed germination of P.
   japonicus.

Fig. 7.

   [121]Fig. 7
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   Heat map of DEGs related to phenylpropanoid biosynthesis. The relative
   expression levels of DEGs were calculated using the Log[2]FC

Transcription factors

   A total of 9742 TFs were annotated in the transcriptome of P. japonicus
   seeds and classified into 58 families (Table [123]S10). Most of the TFs
   were belonged to the bHLH, MYB_related, ERF, NAC, C2H2, FAR1, C3H,
   bZIP, MYB, WRKY, B3 and GRAS families (Fig. [124]8A). Compared with the
   DS, 1819 and 2880 TFs were differentially expressed in the RS and GS,
   respectively. The majority of differentially expressed TFs, which
   belonged to the fourteen families, were significantly upregulated in
   the comparisons of “DS_vs_RS” (Fig. [125]8B), “DS_vs_GS”
   (Fig. [126]8C), and “RS_vs_GS” (Fig. [127]8D). Moreover, most of the
   genes in bHLH, MYB, WRKY, and bZIP families were significantly
   upregulated in both RS and GS, compared with the DS (Fig. [128]8E).
   Thus, we supposed that these continuously upregulated TFs might play a
   key role in the seed germination of P. japonicus.

Fig. 8.

   [129]Fig. 8
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   Analysis of transcription factors (TFs) involved in P. japonicus seed
   germination. A. Distribution of TF families. B-D. The expression
   profiles of TFs in the comparison of “DS_vs_RS”, “DS_vs_GS”, and
   “RS_vs_GS”. E. Differentially expressed TFs in different germination
   stages

Validation of the DEGs by qRT-PCR

   To verify the accuracy of RNA-Seq results, ten DEGs related to
   different metabolic pathways were selected for qRT-PCR validation
   (Fig. [131]9A, Table [132]S11). The expression levels of these genes
   were calculated using the 2^−ΔΔCt method, and the results were compared
   with RNA-Seq data. All the data in both RNA-seq and qRT-PCR were
   adjusted to ensure the average value of gene expression levels in DS
   was 1 (Table [133]S11), so as to reduce the gap of the data between
   RNA-seq and qRT-PCR and more intuitively compare the data within them.
   As expected, a positive correlation coefficient (R^2 = 0.5364,
   p < 0.0001) was obtained between the results of RNA-Seq and qRT-PCR
   through linear regression analysis (Fig. [134]9B), which confirmed that
   the RNA-Seq data were reliable and accurate.

Fig. 9.

   [135]Fig. 9
   [136]Open in a new tab

   Verification of DEGs in transcriptome by qRT-PCR. A. Comparison of the
   gene expression levels determined by qRT-PCR and RNA-Seq. B.
   Correlations between RNA-Seq and qRT-PCR data. All data in the
   histogram are presented by mean ± SD

Discussion

Changes of physiological and biochemical indexes during P. japonicus seed
germination

   Generally, the respiratory metabolism will enhance with the germination
   process and produce a large number of reactive oxygen species (ROS),
   which results in the accumulation of superoxide anion (O[2]^−),
   hydrogen peroxide (H[2]O[2]), and hydroxyl radicals (OH^−) [[137]10].
   These ROS have strong oxidative capacity, which can cause lipid
   peroxidation and oxidative damage to cellular structure, thus promoting
   the accumulation of MDA [[138]21]. During the seed germination of
   Cinnamomum migao, the MDA levels in the LK stage (seed coat fissure
   after imbibition for 24 days) and MF stage (radicle protruding the seed
   coat (4 mm) after imbibition for 31 days) were significantly higher
   than that in the GZ stage (seeds without absorbing water) [[139]10].
   MDA is the byproduct of damaged cell membrane induced by excessive ROS
   [[140]22]. The ROS scavenging enzymes like SOD, POD, and CAT, are
   important substances which can effectively protect plants from
   oxidative damage [[141]23]. In this study, the SOD, POD, and CAT
   activities were significantly higher in the germinated seeds (GS) than
   those in the dormant seeds (DS) (Fig. [142]2), indicating that the cell
   structure was subjected to oxidative stress, but the timely clearance
   of ROS by antioxidant enzymes ensured favorable seed germination.
   Notably, the POD activity was nearly 7-fold higher in the GS than that
   in the DS, which was similar to the results observed in Cyclobalanopsis
   chungii during seed dormancy release and germination [[143]24]. The
   previous studies have confirmed that POD has positive effects on seed
   germination through alleviating the oxidative stress [[144]25].
   Therefore, we believe that wet sand storage (25℃) may promote the seed
   germination by activating the ROS scavenging enzymes, and despite
   further verification is needed.

Plant hormone signal transduction involved in P. japonicus seed germination

   Plant hormones regulate plant seed dormancy and germination internally
   through antagonistic and synergistic interactions [[145]6]. Plant seed
   germination was driven by a variety of hormonal signal transduction
   pathways (gibberellin, auxin, cytokinine, salicylic, and
   brassinosteroid) associated with seed germination, cell division, cell
   enlargement, plant growth, and defense system [[146]26]. However, ABA
   and ethylene signaling pathways always show an opposite effect on plant
   seed germination [[147]26]. ABA suppresses both embryo growth and
   endosperm degradation, resulting in the dormancy of seed and delayed
   germination [[148]27]. Similar to our previous results, the expression
   of DEGs associated with ABA synthesis, such as PP2C and PYR/PYL genes,
   were significantly downregulated in the germinated seeds, compared with
   the dormant seeds [[149]16]. DELLA protein is a negative regulator in
   the GA signaling pathway, while GID1 is a GA receptor which
   participates in the degradation of DELLA protein [[150]28]. In the
   present work, the genes encoding DELLA were remarkably downregulated in
   the RS and GS, indicating that wet sand storage could decrease the
   negative regulators in GA signal transduction pathways, thus promoting
   the seed germination of P. japonicus. However, most of the genes
   encoding GID1 were downregulated in both RS and GS. We supposed that
   the degradation of DELLA was occurred before stage II (sand storage for
   60 days, RS), then the low levels of DELLA had negative-feedback
   effects on the accumulation of GID1. Moreover, the expression levels of
   auxin-related DEGs like SAUR, AUX/IAA, and GH3 genes, were
   significantly higher in the germinated seeds than those in the dormant
   seeds (Fig. [151]4). Thus, we speculated that the downregulation of
   PP2C, PYR/PYL, and DELLA and the upregulation of SAUR, AUX/IAA, and GH3
   might play a key role in promoting the seed germination of P.
   japonicus.

   Previous studies reported that brassinosteroid (BR) play a crucial role
   in promoting plant seed germination [[152]29–[153]30]. Genetic and
   transgenic studies demonstrated that the BR-signaling kinases (BSKs)
   represent a small family of kinases which activate BR signaling
   downstream of BRI1 [[154]31]. In addition, BZR1/2 is a transcription
   factor which can promote the expression of genes related to cell
   division and growth [[155]32]. Furthermore, CYCD3 gene regulates cell
   division, cell expansion, and is necessary to promote the cambial cell
   cycle [[156]33]. The TCH4 gene encoding xyloglucan endotransglycosylase
   (XTH) can relax the cell wall and make the cell elongate [[157]34]. In
   our study, the expression of BSK, BZR1/2, CYCD3, and TCH4 genes were
   significantly higher in the GS than those in the RS, which explained
   the phenomenon that the radicle broke through the seed coat in the GS,
   while not in the RS.

Energy supply for P. japonicus seed germination

   Seed germination is dependent on the degradation of starch and sucrose
   in mature seeds, and the sugars from starch hydrolysis are the major
   source of energy for seed germination [[158]35]. α-amylase (AMY),
   α-glucanotransferase (DPE), and α-glucosidase (AGLU) are the major
   enzymes that convert starch to oligosaccharides or glucose [[159]10].
   Our results demonstrated that most of the genes encoding AMY, DPE, and
   AGLU were significantly upregulated in the germinated seeds of P.
   japonicus (Fig. [160]5), indicating that the upregulation of AMY, DPE,
   and AGLU genes in the GS might result in an accelerated degradation of
   starch. Sucrose synthase (SUS) plays a pivotal role in sucrose
   biosynthesis [[161]8]. Beta-glucosidase (BGL) is a ubiquitous enzyme,
   which hydrolyzes β-D-glucosidic bonds of various compounds comprising
   of alkyl-β-D-glucosides, aryl-β-D-glucosides, cyanogenic glucosides,
   disaccharides and short chain oligosaccharides liberating glucose from
   their nonreducing end [[162]36]. In the present study, multiple genes
   encoding SUS were significantly downregulated, while the BGL genes were
   significantly upregulated in the germinated seeds (Fig. [163]5), thus
   indicating the biosynthesis of sucrose was inhibited and the sucrose
   degradation was stimulated, so as to provide enough energy for P.
   japonicus seed germination.

   Plant seed germination requires a considerable amount of energy to
   perform physiological activities. The necessary energy required during
   seed germination mainly comes from the degradation of storage
   substances [[164]37]. Nevertheless, owing to the lack of oxygen, the
   initial energy supply for the physiological activities is mainly
   provided by glycolysis and alcohol fermentation [[165]38], after which
   ATP synthesis is synthesized through the pyruvate metabolism, TCA cycle
   and mitochondrial electron transport [[166]16]. Notably, the multiple
   enzymes like phosphoglucomutase (PGMC), galactose mutarotase (GALM),
   glyceraldehyde-3-phosphate dehydrogenase (G3PC), and pyruvate kinase
   (KPY) are the key limiting enzymes in the glycolysis pathway. In this
   study, most of the genes encoding PGMC, GALM, G3PC, and KPY were
   significantly upregulated in the RS and GS, compared with the DS, and
   the expression levels of HXK, PFKA, ALF and PGK were relatively higher
   in the RS than those in the DS and GS, indicating the seeds in the
   ringent stage (RS) required more energy than those in the DS and GS.
   These results were not consistent with our previous study [[167]16],
   which indicated that the Michelia chapensis seeds in the advanced stage
   (similar to GS) required more energy than those in the intermediate
   stage (similar to RS), this may be attributed to the diverse energy
   requirements of different plant seeds in different germination stages.
   Generally, pyruvate plays the role of an intermediate product and is
   converted to acetyl-CoA by ACSA and other enzymes [[168]39]. In this
   study, KEGG annotation analysis identified ODP and MDH as a group of
   rate-limiting enzymes in the pyruvate metabolism or TCA cycle. The
   expression levels of ODP and MDH are significantly upregulated in the
   RS, compared with the DS, indicating the wet sand storage (25℃) can
   accelerate the pyruvate metabolism and TCA cycle by up-regulation of
   these genes to provide enough energy for the seed germination of P.
   japonicus. Oxidative phosphorylation is the final metabolic pathway of
   cell respiration and plays an important role in electron transfer and
   energy supply in various metabolic processes [[169]40–[170]41]. In the
   present study, most of the genes involved in oxidative phosphorylation
   pathway were significantly upregulated in the stage “from DS to RS”,
   while downregulated in the stage “from RS to GS”, indicating the seeds
   required a large amount of energy for the development of embryo in the
   initial germination stage of P. japonicus (from DS to RS), and
   relatively less energy was required for the seed germination after the
   embryo development was basically completed and the radicle broke
   through the seed coat (from RS to GS).

Phenylpropanoid biosynthesis related to P. japonicus seed germination

   In higher plants, the phenylpropanoid biosynthesis is associated with
   the seed antioxidant capacity during germination [[171]42]. Reportedly,
   the fluctuations of PAL levels are thought to be a key factor in the
   control of phenylpropanoid biosynthesis [[172]43]. PAL can catalyze the
   nonoxidative deamination of [L]-Phe and yield t-CA in plants [[173]44].
   4CL controls phenylalanine in different metabolic pathways. COMT can
   catalyze the methylation of caffeic acid to produce ferulic acid and
   other intermediates, which can be further converted into CoA ester as a
   precursor for lignin and flavonoid synthesis [[174]45]. Moreover, PAL,
   4CL, and COMT are the key rate-limiting enzymes of the whole metabolic
   pathway, which directly determine whether various secondary metabolic
   pathways can proceed smoothly [[175]46]. Generally, POD acts as an
   antioxidant and participates in the peroxide degradation, thereby
   effectively eliminating the damage caused by free radicals on the cell
   membrane [[176]47]. In the present study, we found that the PAL, 4CL,
   COMT, and POD genes were significantly upregulated in the GS, compared
   with the DS, which was similar to the results observed in the seed
   dormancy release and germination processes of Cinnamomum migao
   [[177]10] and Michelia chapensis [[178]16], indicating the antioxidant
   capacity was increased in the germinated seeds, compared with the
   dormant seeds. These results were consistent with the higher
   antioxidant enzyme activities in the germinated seeds (Fig. [179]2).
   Hence, we speculated that these DEGs mentioned above played an
   important role in releasing P. japonicus seed dormancy by regulating
   phenylpropanoid biosynthesis.

Candidate TFs associated with P. japonicus seed germination

   The gene expression pattern regulated by TFs plays crucial roles in
   plant growth and seed germination [[180]48]. MYB, bHLH, bZIP, and WRKY
   are the major categories families of transcription factors related to
   plant growth and seed germination [[181]49]. The MYB is a major
   superfamily of TFs which regulates plant growth, seed germination, and
   the biosynthesis of primary and secondary metabolites [[182]50]. In
   Arabidopsis seeds, the MYB44 gene is upregulated by 4 °C treatment and
   acts as a negative regulator of ABA signaling [[183]51]. Likewise, bHLH
   is the second largest transcription factor family and play an important
   role in regulating plant growth and development [[184]52–[185]53]. bZIP
   TFs control various biological processes, such as pathogen defense,
   light and stress signals, seed maturation, and flower development
   [[186]54]. The bZIP16 gene is mainly expressed in seeds and can
   stimulate the GA pathway and inhibit ABA action, thereby promoting seed
   germination [[187]55]. WRKY TFs are involved in various plant
   activities, such as development and metabolism, growth and senescence,
   and responses to biotic and abiotic stresses [[188]56–[189]58].
   Interestingly, we found that most of the TFs in MYB (CSLA2, CSLA9,
   MYB61, XTHB), bHLH (PAE8, EXPA4, EXPA6, MYC3), bZIP (PAN, ARAC9, TGA1),
   and WRKY (UGD1, NAT6), were significantly upregulated in both RS and
   GS, compared with the DS (Fig. [190]8E). Similar results were observed
   in the seed germination of Michelia chapensis [[191]16]. Consequently,
   we presumed that these continuously upregulated TFs played a crucial
   role in regulating P. japonicus seed germination.

Conclusion

   In this study, sand storage at 25 ℃ (65% relative humidity) for 120 d
   effectively released the seed dormancy of P. japonicus and promoted the
   seed germination up to 89%. The activities of the SOD, POD, and CAT
   were significantly increased in the germinated seeds (sand storage for
   120 days, GS), compared with the dormant seeds (no sand storage, DS),
   indicating higher antioxidant capability of P. japonicus seeds in the
   GS than in the DS. The seed germination of P. japonicus was regulated
   by the synergies of biological pathways like plant hormonal signal
   transduction, starch and sucrose metabolism, energy supply (glycolysis,
   pyruvate metabolism, and oxidative phosphorylation), phenylpropanoid
   biosynthesis, antioxidant metabolism, and transcriptional regulation
   (Fig. [192]10). In addition, the biological events which occurred at
   the transcriptional level were consistent with the phenotype,
   physiological indexes, and qRT-PCR results. This study contributes to
   improving the propagation efficiency and provides crucial information
   regarding the potential mechanisms of P. japonicus seed germination.

Fig. 10.

   [193]Fig. 10
   [194]Open in a new tab

   A proposed model showing the changes of biological events occurred in
   the P. japonicus seed germination. The genes or physiological indexes
   labeled with red were upregulated, while labeled with green were
   downregulated

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [195]Supplementary Material 1^ (1MB, docx)
   [196]Supplementary Material 2^ (331KB, xlsx)

Acknowledgements