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Article

Transcriptome Analysis Reveals the Effects of Exogenous Gibberellin on the Germination of Solanum torvum Seeds

by
Saimei Wu
1,2,†,
Qidong Si
1,2,†,
Kejie Yang
1,2,
Wenwei Zhang
1,2,
Laining Zhang
1,2,
Thomas W. Okita
3,
Yanyan Yan
1,2,* and
Li Tian
1,2,*
1
Collaborative Innovation Center for Efficient and Green Production of Agriculture in Mountainous Areas of Zhejiang Province, College of Horticulture Science, Zhejiang A&F University, Hangzhou 311300, China
2
Key Laboratory of Quality and Safety Control for Subtropical Fruit and Vegetable, Ministry of Agriculture and Rural Affairs, Zhejiang A&F University, Hangzhou 311300, China
3
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA
*
Authors to whom correspondence should be addressed.
†
These authors contribute equally to this work.
Agronomy 2024, 14(8), 1736; https://doi.org/10.3390/agronomy14081736
Submission received: 23 July 2024 / Revised: 3 August 2024 / Accepted: 6 August 2024 / Published: 7 August 2024
(This article belongs to the Special Issue Effect of Agronomic Treatment on Seed Germination and Dormancy)
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Abstract

:
Solanum torvum, a wild relative of eggplant in the Solanaceae family, is widely used as a rootstock to prevent various soil-borne diseases. Despite this valued trait, S. torvum seeds exhibit strong seed dormancy, resulting in low-frequency germination and uneven emergence rates during agricultural production. In this study, exogenous gibberellin (GA) was applied to induce the germination of seeds, thereby enabling the application of transcriptome analysis to explore the effects of exogenous GA on germination. A total of 9723 genes were identified to be involved in GA-induced germination of S. torvum seeds. These GA-associated genes were enriched in amino sugar and nucleotide sugar metabolism, glycolysis, the citrate cycle, pyruvate metabolism, hormone biosynthesis, and signaling transduction. Further analysis of these genes reveals that exogenous GA impaired the endogenous biosynthesis of GA. Analysis of the 285 key genes influenced by GA, auxin, abscisic acid (ABA), and other hormones suggests mutual crosstalk in the signaling of various hormones, enabling the joint regulation of the germination of S. torvum seeds. Additionally, a total of 440 genes encoding transcription factors were identified to be responsible for transcriptional reprogramming during GA-induced seed germination, and putative candidate genes responsible for S. torvum seed dormancy and germination, including ABI5, DOG1, DRM1, LEC1, and PIF, were significantly down-regulated in germinated S. torvum seeds. These findings provide the first insights into transcriptome regulation during GA-induced germination of S. torvum seeds.

1. Introduction

Eggplant (Solanum melongena L.) is a popular vegetable crop because of its preferred flavor and high nutrition. According to the data provided by the World Food and Agriculture Organization (FAO) in 2023, eggplant is one of the most cultivated vegetable crops, attaining global production of 59 million tonnes [1]. Eggplants are susceptible to several soil-borne diseases, including bacterial wilt, fusarium wilt, and verticillium wilt [2]. Along with the continuous expansion of the eggplant cultivation area, soil-borne diseases have become more frequent due to repeated cropping as well as increased temperature and high humidity [3]. Once the bacteria or fungal pathogens infect the host roots and invade the vascular system, the host plants exhibit wilt-symptomatic vessels [4], which eventually causes plant death and, in turn, severe production loss worldwide.
To prevent vascular wilt diseases, eggplants are usually grafted onto resistant rootstocks [5,6,7]. The commonly used rootstock is Solanum torvum Swartz., a wild relative of eggplant in the Solanaceae family. S. torvum is a native shrub that originates from tropical regions of Africa and Asia and is widely found in scrub, roadside, wasteland, or damp places. The species shows high resistance to bacterial and fungal wilt [8] and various environmental stresses, such as salt and cadmium stresses [9,10]. It is, therefore, widely employed in agricultural production as a rootstock for grafting other solanaceous plants, such as tomato, eggplant, and pepper, to improve plant growth, yield, and resistance to diseases and pests [11,12,13].
Due to the strong dormancy, the germination of S. torvum seeds is poor and usually requires considerable time [14], which causes uneven emergence during S. torvum rootstock production. Several methods, such as the application of low temperature, soaking treatment in K3PO4, KNO3, and NaNO3, and the application of exogenous gibberellin (GA), have been applied to break seed dormancy and promote S. torvum seed germination [14,15,16]. Among these methods, the application of GA is the most effective treatment, which breaks seed dormancy and significantly improves the seed germination rate [14,15,16].
GA is a key phytohormone that regulates seed dormancy and germination. It stimulates seed germination by breaking seed dormancy and promotes embryo–seedling transition by turning over seed inhibitors in the seed and activating the synthesis of enzymes that are necessary for germination [17,18,19]. Consistent with the view that elevated GA levels are responsible for dormancy loss in wheat and barley [20,21,22], Arabidopsis and tomato seeds carrying mutations in GA biosynthetic enzymes exhibited a defect in germination [23,24,25]. For example, Peng and Harberd [26] found that tomato seeds lacking GA (gib-1) could not germinate without exogenous GA. These results suggest that the level of GA plays a crucial role in breaking seed dormancy and enabling seed germination. Thus, exogenous application of GA has been widely applied to break seed dormancy and facilitate seed germination.
Although the impact of GA on seed germination has been widely studied in many plant species [27,28,29,30], no study has been undertaken to investigate the underlying mechanism by which exogenous GA stimulates S. torvum germination. In this study, transcriptome analysis was performed to investigate the regulation of gene expression during GA-induced germination of S. torvum seeds. Such effort identified potential genes that are induced by GA and thereby regulate plant energy metabolism and hormone signaling transduction to promote the seed germination process.

2. Materials and Methods

2.1. Plant Materials and Seed Germination

Seeds from S. torvum were obtained from Will Seed Company (Jinan, Shandong, China) and used for a germination test. The seeds were first incubated in 1.5 mM bioactive gibberellin GA3 or distilled water for 24 h at room temperature in the dark. After rinsing 5 times with distilled water, the seeds were placed on two layers of filter paper in Petri dishes and incubated in a growth chamber at 28 °C in darkness. During the germination test, the filter paper was kept moist at all times. Seed germination was monitored for 14 days. When the radicle emerged from the seed coat and extended more than 0.5 cm, the seed was considered to be germinated [31,32]. Germination rates and standard deviations were calculated based on five independent experiments. The germination percentage and the germination index were calculated with the following equations [33]:
Germination   percentage = N u m b e r   o f   g e r m i n a t e d   s e e d s N u m b e r   o f   t o t a l   s e e d s × 100
Germination   Rate   Index   ( GRI ) = ∑ N u m b e r   o f   g e r m i n a t e d   s e e d s   a t   d a y   n n .

2.2. Seed Samples for Transcriptomic Analysis

To ensure all the germinated (G) seeds used for transcriptome analysis were uniform, the seeds were collected immediately once their radicles extended to 0.5 cm. After 12 days, the seeds from the GA-treated group without the emergence of the radicles were collected as ungerminated (NG) seeds. No seeds treated with distilled water for 12 days were observed to be germinated, and were thereby used as a control group (W). Three biological replicates were performed for each treatment.

2.3. Transcriptomic Analysis

Total RNA was extracted from each sample using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and RNA integrity was analyzed using an Agilent 2100 bioanalyzer. The mRNA obtained by Oligo(dT) magnetic beads was used for library construction using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) according to the manufacturer’s protocol. Initial quantification of the constructed library was performed using Qubit 2.0. After passing the library inspection, the libraries were sequenced on Illumina Novaseq X. After removing low-quality reads, the remaining high-quality reads were mapped against the recently sequenced genome of S. torvum [34].

2.4. Identification of DEGs

Raw data of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of all recognized genes are presented in Supplemental Table S3. Based on the expression level of FPKM, genes with a log2 fold change > 1 and a p value < 0.01 were defined as differentially expressed genes (DEGs) (Supplemental Table S6). ClusterProfiler software (Version: 3.18.0) was used to analyze the enrichment of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms. GO and KEGG terms with p values < 0.05 were considered to be significantly enriched. Significant differences (p value) were calculated by Student’s t-test.

3. Results

3.1. Effect of GA Treatment on Seed Germination of S. torvum

In our preliminary experiment (Supplemental Figure S1), no germination of S. torvum seeds was observed when treated at low temperature (4 °C) or when pre-soaked in KNO3 (20 g/L), MgSO4 (20 g/L), or CaCl2 (20 g/L). Exogenous application of GA treatment, however, stimulated the germination of S. torvum seeds (Supplemental Figure S1). Among the various concentrations of GA, 1.5 mM was the minimum concentration to effectively promote S. torvum seed germination (Supplemental Figure S1). Thus, treatment with 1.5 mM GA was used for the subsequent analysis. After 24 h pre-treatment of 1.5 mM GA, around 10% of GA-treated S. torvum seeds germinated at day 5. Eight days after treatment, the germination rate reached 70%. Although slow germination was still observed after day 8, the peak of germination (around 85%) and the highest Germination Rate Index were observed at day 11 (Figure 1A). By contrast, no germination of S. torvum seeds was observed under pre-treatment with H2O (Figure 1A). Based on these results, pre-soaking treatment with 1.5 mM GA was used to induce the germination of S. torvum seeds, and H2O treatment was used as a negative control.

3.2. Identification of Differentially Expressed Genes (DEGs) between Germinated and Ungerminated S. torvum Seeds

To investigate the germination mechanism of S. torvum seeds, RNA-seq analyses on a total of nine samples from germinated and ungerminated seeds were performed (Supplemental Figure S2). Three RNA samples, G1, G2, and G3, were extracted from the germinated seeds after GA treatment, while three RNA samples of GA-treated ungerminated seeds were labeled as NG1, NG2, and NG3. The remaining three samples were prepared from H2O treatment (W) as control.
A total of 30,543 genes, including 4842 novel genes, were detected in the nine samples (Supplemental Tables S2–S4). Among them, 26,526 and 25,944 genes were detected in W and NG samples, respectively, while 28,119 genes were detected in G samples. This result suggests that the transcriptome was more active in germinated seeds when compared to water-treated and GA-treated ungerminated seeds. Principal component analysis (PCA) and correlation analysis (Supplemental Figure S3, Table S5) revealed that after GA pre-treatment, the transcriptome of NG seeds was highly related to that in group W, while the transcriptome in group G showed a large variation from groups W and NG.
Compared to the H2O-treated control, 12,593 and 1626 genes were differentially expressed genes in the germinated (G vs. W) and ungerminated seeds (NG vs. W), respectively (Figure 2A, Supplemental Table S6). The number of up-regulated genes in the G group was 7142, which was more than the down-regulated genes (5451). The numbers of up- and down-regulated genes in the NG group were 784 and 842, respectively. In the comparison between groups G and NG (G vs. NG), 11,572 DEGs, including 6576 up- and 4996 down-regulated genes, were detected. A heatmap cluster analysis (Figure 2B) further revealed that most of the DEGs showed similar expression patterns in W and NG when compared to the G group. All these results suggested that the transcriptome was significantly reprogrammed in germinated seeds, while only a small number of genes in S. torvum seeds were induced in GA-treated ungerminated seeds.
KEGG enrichment analysis of DEGs (Figure 2C) revealed that pathways of carbon metabolism, the citrate cycle (TCA cycle), carbon fixation in photosynthetic organisms, and the biosynthesis of amino acids were significantly enriched in DEGs detected in both NG- and G-group seeds, suggesting that GA pre-treatment induced a change in energy metabolism and amino acid biosynthesis. No significant difference in pathway enrichment was observed between the G vs. W and G vs. NG comparisons. The enriched pathways included amino sugar and nucleotide sugar metabolism, the biosynthesis of nucleotide sugars, phagosome, glycolysis/gluconeogenesis, glycoxylate metabolism, and dicarboxylate metabolism and pyruvate metabolism. However, DEGs in the NG vs. W group were highly enriched in ribosome-related synthesis, which was different from that in G vs. W and G vs. NG comparisons. Further analysis of up- and down-regulated DEGs (Supplemental Figure S4) revealed that while up-regulated pathways were similar to that detected in all DEGs, only a few of pathways, such as basal transcription factors and spliceosome, were significantly down-regulated in the G vs. W and G vs. NG groups.
GO enrichment analyses (Supplemental Figures S5–S7) were further performed on each group. DEGs in the G vs. W and G vs. NG groups were enriched in similar GO terms for carbohydrate metabolism, the endomembrane system, and pyrophosphatase activity (Supplemental Figures S5 and S6). With regard to up-regulated GO terms, carbohydrate metabolism-related processes, cytoplasmic part, and structural molecular activity were prominent. On the other hand, GO terms related to membrane-bound organelle and RNA metabolic pathways were significantly down-regulated. In the NG vs. W group (Supplemental Figure S7), however, GO terms of non-membrane-bound organelle and small molecular metabolic processes were highly up-regulated, while DNA repair-related GO terms were detected in down-regulated DEGs.

3.3. Identification of Germination-Related Genes (GRGs)

Venn diagrams revealed that only 1206 common genes were detected between the NG vs. W and G vs. W groups (Figure 3A,B). Those genes may be directly induced by GA pre-treatment. On the other hand, a total of 9723 DEGs (Supplemental Table S7), including 673 commonly regulated genes in seeds pre-treated by GA and 9050 specifically regulated genes in germinated seeds, were identified to be germination-related genes (GRGs).
KEGG and GO enrichment analyses (Figure 3C,D) were performed on those GRGs. The pathways of amino sugar and nucleotide sugar metabolism, the biosynthesis of nucleotide sugars, the citrate cycle (TCA cycle), carbon metabolism, and pyruvate metabolism were profoundly enriched (Figure 3C), suggesting that genes related to energy metabolism were significantly regulated during germination. On the other hand, GO terms related to pyrophosphatase activity, hydrolase activity, and nucleoside-triphosphatase activity were significantly prominent, which supported the KEGG enrichment of energy metabolism. In addition, GRGs related to photosynthesis and the cytoskeleton were also over-represented in KEGG and GO enrichment analyses.

3.4. GRGs Related to Energy Metabolism in S. torvum Seed Germination

Given that energy-related metabolic pathways were highly enriched during the germination of S. torvum seeds, the GRGs related to amino sugar and nucleotide sugar metabolism, glycolysis, the citrate cycle (TCA cycle), and pyruvate metabolism were further analyzed (Figure 4, Supplemental Table S8). Expression profiling analysis on 122 GRGs encoding the key enzymes involved in these pathways indicated that most of them were up-regulated in germinated seeds (Figure 4). The expression of eight genes encoding endochitinase (CHIT), two genes encoding hexokinase (HXK), and two genes encoding UTP-glucose-1-phosphate uridylyltransferase (G6PI), with the latter two enzymes promoting the biosynthesis of Fru-6-P, was significantly enhanced in germinated seeds. Fru-6-P is the key substrate of ATP-dependent 6-phosphofructokinase (PFK) for the synthesis of the metabolite regulator, Fru-1,6-P2, which is eventually converted into pyruvate in the glycolysis process. Most of the genes encoding the key glycolytic enzymes, including PFK and pyruvate kinase (PK), were up-regulated. Up-regulated expression was also detected in the genes encoding the key enzymes involved in pyruvate metabolism, including NAD-dependent malic enzyme (ME), phosphoenolpyruvate carboxylase (PEPC), pyruvate decarboxylase (PDC), pyruvate dehydrogenase (PDH), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH). The expression of 22 TCA cycle genes encoding citrate cleavage-related enzymes, such as ATP-citrate synthase (ACYL), malate dehydrogenase (MDH), succinate dehydrogenase (SDH), and dihydrolipoyl dehydrogenase (DLD), was generally up-regulated in germinated seeds. These results suggest that energy-related metabolism is enhanced during GA-induced seed germination.

3.5. GRGs Related to Hormone Biosynthesis and Signaling Transduction during S. torvum Seed Germination

Although genes related to the hormone signaling transduction pathway were not detected to be highly enriched in DEGs or GRGs, several key factors involved in the hormone signaling transduction pathway were observed to be significantly influenced during GA-induced seed germination (Supplemental Figure S8). We then further analyzed the expression profile of GRGs that are related to the hormone biosynthesis and signaling transduction pathways (Supplemental Table S9). Among the 260 GRGs related to hormone biosynthesis and signaling, the major contribution came from GA, abscisic acid (ABA), auxin, and ethylene.
Whether exogenous application of GA affected the expression of genes involved in the GA biosynthesis and signaling pathway was firstly analyzed (Figure 5). GA biosynthesis in higher plants (Figure 5A) involves production of ent-kaurene in proplastids, the conversion of ent-kaurene to bioactive GA1, and the synthesis of various forms of GAs in the cytoplasm [31,32]. The GA signal is captured by the GA INSENSITIVE DWARF1 (GID1) receptor. The binding of GA with GID1 promotes the interaction of GID1 with DELLA proteins that mediates the degradation of DELLAs via the ubiquitin–proteasome pathway [35,36]. Thus, the transcriptional repression of GA response genes by DELLAs is released, which, in turn, induces GA signaling transduction. The expression of several genes encoding GA biosynthesis-related key enzymes such as gibberellin 20-oxidase (GA20ox), gibberellin 2-beta-dioxygenase (GA2ox), gibberellin 3-oxidase (GA3ox) and gibberellin 13-oxidase (GA13ox) was transcriptionally regulated (Figure 5B). Among these genes, Sol0149840 encoding GA20ox and Sol0085000 encoding GA13ox were significantly down-regulated, while Sol0164590 encoding GA3ox was greatly up-regulated. Most of the detected genes encoding GA2ox were transcriptionally enhanced. Several genes encoding key factors involved in GA signaling, including GID1, DELLA, and GA-responsive transcription factors (TFs), were transcriptionally regulated as well. While a GID gene was detected to be down-regulated, a gene (Sol0088980) that encodes the DELLA protein was found to be significantly up-regulated. Most GA-responsive TFs were repressed. These results suggest that exogenous GA treatment disturbed the endogenous GA biosynthesis and signaling transduction.
A total of 51 genes were identified to be involved in the ABA biosynthesis and signaling pathway. Xanthoxin, the intermediate metabolite during ABA biosynthesis, is synthesized from β-carotene (Figure 6A). The relative key enzymes, however, including beta-carotene hydroxylase (BCH), zeaxanthin epoxidase (ZEP), and 9-cis-epoxycarotenoid dioxygenase (NCED), were largely down-regulated (Figure 6B, Supplemental Table S9).
Core components in ABA signaling involve the receptor PYR/PYL, the negative regulator PP2C, the positive regulator SnRK, and the downstream responsive TFs. ABA activates its receptors, PYR/PYL family proteins, to inhibit the activity of PP2Cs on SnRKs, which activates SnRKs to stimulate the ABA response [37]. While most of the identified GRGs encoding PYL and SnRKs were transcriptionally up-regulated in germinated seeds, both up- and down-regulation of PP2C coding genes were observed (Figure 6B). Some ABA-responsive TFs, such as ABSCISIC ACID INSENSITIVE5 (ABI5), G-box binding factor (GBF), and Transcription factor responsible for ABA regulation 1 (TRAB1), were found to be down-regulated. These results suggest that ABA biosynthesis and signaling may be repressed during GA-induced seed germination.
The expression patterns of GRGs involved in auxin biosynthesis and signaling are shown in Figure 7. Auxin is mainly produced via a tryptophan-dependent pathway. Tryptophan, as the auxin precursor, is synthesized from chorismate via the shikimate pathway (Figure 7A). A total of 19 genes encoding auxin biosynthetic enzymes, including flavin-containing monooxygenase (YUCCA), L-tryptophan–pyruvate aminotransferase (TAA), anthranilate phosphoribosyltransferase (AnPRT), anthranilate synthase (ANS), amidase (AMI), aldehyde dehydrogenase (ALDH), and tyrosine decarboxylase (TDC), were found to be transcriptionally regulated. While the expression of most of these genes was significantly enhanced (Figure 7B), one AMI-coding gene and two ALDH-coding genes were among the down-regulated GRGs. Auxin transport is carrier-mediated. AUXIN1/LIKE-AUX1 (AUX1/LAX) family members function as auxin influx carriers, while PIN-FORMED (PIN) family members are the major auxin efflux carriers [38]. The expression of six genes encoding LAX and PIN family proteins was observed to be enhanced in germinated seeds (Figure 7C). Three core signaling components, including the receptors TIR1/AFBs, the transcriptional repressors Aux/IAAs, and the transcription factors ARFs, mediate the transcriptional response to auxin in the nucleus [39,40,41,42,43]. A large number of genes encoding the core components involving auxin signaling, including TIR1, AUX/IAA, ARF, small auxin up-regulated RNA (SAUR), and GRETCHEN HAGEN 3 (GH3), were found to be transcriptionally regulated in germinated seeds (Figure 7C). While the majority of early auxin responsive genes like AUX/IAA, SAUR, and GH3 were found to be transcriptionally enhanced, TIR1-coding genes were significantly repressed. These results suggest that auxin biosynthesis and signaling may play a role in response to GA-induced seed germination.
Another enriched hormone signaling pathway in GRGs is ethylene. Ethylene is synthesized from L-methionine by three key steps catalyzed by S-adenosylmethionine synthase (SAMS), 1-aminocyclopropane-1-carboxylate synthase (ACS), and 1-aminocyclopropane-1-carboxylate oxidase (ACO) (Figure 8A). As shown in Figure 8B, most of the genes encoding SAMS, ACS, and ACO were significantly up-regulated, suggesting an enhancement of ethylene biosynthesis in GA-induced germinated seeds. Core components in ethylene signaling, such as ethylene receptors (ETRs), constitutive triple response 1 (CTR1), ethylene-insensitive 2 (EIN2), ethylene-insensitive 3 (EIN3), and ethylene response factors (ERFs), were also analyzed (Figure 8B). Ethylene functions as an inverse agonist to its receptor ETR [44], which releases the inhibitory activity of CTR1 on the downstream ethylene responsive transcription. While the expression of the identified ETR and CTR genes were found to be significantly decreased in germinated seeds, most of the ethylene-responsive transcription factors EIN3 (Figure 8B) and ERF (Figure 8C) were activated. All these results suggest that both ethylene biosynthesis and signaling pathways were enhanced during GA-induced seed germination.
The genes encoding jasmonate (JA) and salicylic acid (SA) signaling-related components, such as jasmonic acid-amido synthetase (JAR1) and JASMONATE ZIM-DOMAIN (JAZ) in JA signaling and nonexpressor of pathogenesis-related 1 (NPR1), transcription factor TGA1, and salicylic acid-binding protein 2 (SABP) in SA signaling, were mostly up-regulated (Supplemental Table S9). Most A-type ARABIDOPSIS RESPONSE REGULATOR (ARR) family members that mediate the cytokinin signaling response were up-regulated, while differential expression of brassinosteroid (BR)-insensitive 1 (BRI1) and BR-regulated transcription factor BZR1 in BR signaling was also observed (Supplemental Table S9). Collectively, these results suggest that hormone signaling pathways are significantly influenced during GA-induced seed germination.

3.6. Possible TFs Responsible for Seed Germination

A total of 440 TFs were identified in GRGs (Figure 9, Supplemental Table S10). Among those TFs, 79 TFs belong to zinc finger motif-containing factors, 67 MYB family members, and 56 bHLH family members (Figure 9A). Based on their expression in germinated seeds in comparison with ungerminated seeds, the TFs were clustered into two groups. A total of 169 TF coding genes showed decreased expression while the expression of 260 TFs was increased in germinated seeds (Figure 9B,C). These genes may be responsible for transcriptional reprogramming during GA-induced seed germination.

3.7. Genes That Are Responsible for Seed Dormancy and Germination

We further analyzed the expression of known genes encoding the key factors responsible for seed dormancy and germination (Table 1), including Delay of Germination-1 (DOG1), Dormancy-associated DRM1/APR-like protein, FUSCA 3 (FUS3), LEAFY COTYLEDON 1 (LEC1), chromatin-remodeling factor PICKLE (PKL)-related protein, PHYTOCHROME INTERACTING FACTOR1 (PIF1), and REVEILLE 1 (RVE1). Transcriptome data revealed that their expression was decreased during GA-induced seed germination compared to that observed in ungerminated seeds. For example, DOG1-like protein coding gene (Sol0034020.1) was expressed in H2O-treated ungerminated seeds with an average FPKM of 90, which was decreased to 1 in germinated seeds (Supplemental Table S6) with a log2 fold change of −6.5 (Table 1).

4. Discussion

Seed germination is a complex process involving dormancy breaking, embryo recovery from drying, seed coat rupture, radicle elongation, and germ transformation into seedlings [45,46]. The process is regulated by the coordination of different external environment and endogenous signals. A variety of techniques are often used to treat seeds to induce germination. For example, Arabidopsis seeds must be treated at low temperatures to induce germination [47]. Dufková et al. [48] proposed that hydrogen peroxide and temperature treatment improved seed germination. In addition, some physical methods, such as hand-dehulling seeds, can also effectively promote seed germination [49].
GA is an essential plant hormone which is mainly synthesized in buds, young roots, and immature seeds and is involved in many biological processes such as seed germination, flowering, and fruit development [26]. Exogenous treatment of the plant phytohormone GA is effective in promoting seed germination in a variety of crops, such as Arabidopsis [19], rice [50], and Fraxinus hupehensis [51]. In the study of peony, apple, poplar, pear, grape, etc., exogenous spraying of GA was used to replace low temperatures to break seed dormancy [52].
A previous study [14] revealed that although less efficient than GA, application of KNO3 had positive effects in releasing S. torvum seeds from dormancy and stimulating germination. However, in our study, KNO3 was not effective while GA was required to induce the germination of S. torvum seeds. It was suggested that the positive effects of chemical substances, such as KNO3, are not always observed due to their abilities to decrease the osmotic pressure of the substrate, thereby precluding imbibition [15,53]. The germination induced by KNO3 may require further investigation. It is worth mentioning that while GA helped to break the dormancy of S. torvum seeds, seedling emergence was not uneven. Eight days were required to reach the highest germination rate, with about 15% of the seeds remaining ungerminated after 12 days (Figure 1). Similar results were reported in previous studies [14,54]. While a test on multiple factors revealed an emergence rate of only 77% after 14 days [14], the highest germination of 81% was detected when GA treatment was combined with altering temperature [54]. Thus, to further improve the germination of S. torvum seeds for usage as a rootstock and for breeding purposes, the underlying mechanism for efficient germination rates needs to be investigated [7,16].
RNA sequencing allows for a quantitative view of gene expression at the transcript level. The transcriptome reprogramming reflected by RNA sequencing enables the investigation of the alteration of key regulation pathways during a specific biological process. The present study utilizes RNA-seq to investigate the effects of GA on the germination of S. torvum seeds.
Analysis of the detected DEGs and GRGs revealed that energy-producing metabolic pathways, including amino sugar and nucleotide sugar metabolism, the biosynthesis of nucleotide sugars, the citrate cycle (TCA cycle), carbon metabolism, and pyruvate metabolism, were largely elevated during the germination of S. torvum seeds. This result aligns with previous studies showing that glycolysis, the tricarboxylic acid cycle (TCA cycle), and amino acid metabolism play key roles in seed germination [55,56]. The enhancement of carbon metabolism reflects the utilization of stored sugars and starches in seeds during germination [57], which provide a large amount of energy for seed germination. Glycolysis and alcoholic fermentation are induced under limited oxygen content during seed imbibition [58]. They provide additional energy to satisfy the energy demands of germinating seeds. Consistent with this view, several genes encoding the rate-limiting enzymes in glycolysis such as HXK, PFK, and PK, as well as ethanol fermentation related-enzymes such as PDC and ADH, were up-regulated in GA-induced germinated seeds (Figure 4). Likewise, the TCA cycle was promoted in germinated seeds, which produces a large amount of energy (ATPs) and reduces the power (NADH and FADH2) required for seed germination (Figure 4). The rate-limiting enzyme in the TCA cycle, IDH, was also found to be activated (Figure 4). Meanwhile, the expression of genes encoding the vital enzyme, PDH, that links anaerobic glycolysis to the aerobic TCA cycle was largely activated (Figure 4), which boosts pyruvate metabolism to produce a large amount of acetyl-CoA as the major substrate of the TCA cycle. The mobilization of these metabolic pathways is to provide sufficient energy for seed germination. Such metabolic processes were not activated in ungerminated seeds.
The changes in energy metabolism may be regulated by exogenous GA treatment. Duan et al. [59] showed that GA promoted early seed germination by influencing physiological and metabolic activities of seeds. In a study on potato tubers, application of GA was found to induce the activity of α-amylases and β-amylases [60]. GA can also promote the synthesis of nucleic acid and protein by activating hydrolytic enzymes in seeds, thus promoting the germination of plant seeds. It was found that the active Cys peptide sites of reduced glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 acted as redox switches to induced early response during early germination [61]. Their mutations resulted in an enhancement in the oxygen consumption rate and dysregulation of TCA cycle dynamics and significantly reduced the seed germination rate [61]. Therefore, we hypothesize that the changes in energy metabolism-related genes during the germination of S. torvum seeds is caused by exogenous addition of GA.
However, exogenous addition of GA appears to affect endogenous GA biosynthesis and signaling transduction. The genes encoding enzymes involved in early steps of GA biosynthesis, i.e., CPS, KS, KO, and KAO, were not differentially expressed between germinated and ungerminated seeds. On the other hand, however, the enzymes in the third stage of GA biosynthesis, such as GA2ox, GA3ox, GA20ox, and GA13ox, were highly enriched in GRGs (Figure 5). Among them, GA20ox is the major rate-limiting enzyme in GA biosynthesis, which catalyzes the formation of active GAs. Its expression was greatly depressed in the GA-induced germinated seeds (Figure 5B). Meanwhile, germination enhanced the expression of four genes encoding GA2ox (Figure 5B), which diverges early intermediates from the biosynthesis pathway of active GA forms [62]. It was reported that overexpression of GA2ox significantly affected the germination rate of tomato seeds [63]. The depression of the endogenous biosynthesis of active GA might be a feedback inhibition mediated by the exogenous addition of GA. Consequently, GA signaling transduction is depressed, reflected by the down-regulation of a gene encoding gibberellin receptor GID1 and the up-regulation of the transcriptional repressor in GA signaling, DELLA (Figure 5B) [64,65].
Hormones are widely accepted to be vital in the regulation of seed dormancy and germination. GA and ABA are the two classic phytohormones that antagonistically regulate seed dormancy and germination [66]. While GA stimulates seed germination [67,68,69,70], ABA is involved in the establishment and maintenance of dormancy to inhibit seed germination by restricting the availability of energy and metabolites [71,72]. Seeds of the Arabidopsis ga1-3 mutant, which is deficient in GA biosynthesis, are unable to germinate [73]. On the other hand, artificial induction of ABA synthetase NCED6 and NCED9 was found to significantly delay seed germination [74]. Thus, a balance between ABA and GA is critical for the initiation of seed germination. In this study, exogenous GA-induced seed germination caused the down-regulation of more than 30 genes encoding the major enzymes involved in ABA biosynthesis in plastids, including NCED, ZEP, and BCH, suggesting the inhibition of ABA biosynthesis during seed germination. Meanwhile, the expression of a large number of genes coding the core components of ABA signaling, including PYL, PP2C, and SnRK2, were also disturbed in germinated S. torvum seeds (Figure 6).
Auxin is another hormone required for seed development and dormancy. Exogenous auxin regulates the secondary dormancy and germination of tobacco seeds [75]. Auxin is considered to promote seed dormancy and inhibit seed germination by increasing ABA content while impairing the biosynthesis of active GA forms [76,77]. The crosstalk between auxin and ABA is also reflected by the application of fluridone, an inhibitor of ABA biosynthesis, which reverses the auxin-mediated delay in germination [77]. On the other hand, once seed dormancy is broken, auxin is required to promote hypocotyl elongation [78,79]. We noticed that most of the identified genes encoding IAA biosynthetic enzymes were up-regulated (Figure 7), and more than 50 genes encoding the auxin transporters LAXs and PINs and core components in auxin signaling such as AUX/IAA and SAUR were significantly enhanced. These results suggest that auxin biosynthesis and signaling are activated to promote seedling development after the dormancy of S. torvum seeds is broken by exogenous GA.
We also found that the expression of a large number of genes involved in the biosynthesis of other hormones and signaling transduction, including ethylene, cytokinin, BR, JA, and SA, was altered during GA-induced germination of S. torvum seeds. For example, the key enzymes responsible for ethylene biosynthesis from methionine, including SAMS, ACS, and ACO, were largely activated (Figure 8), suggesting that ethylene biosynthesis is elevated in germinating seeds. Ethylene has been reported to play an important role in dormancy release and seed germination in many species [80]. Its regulatory role is considered to engage in crosstalk with other hormones, such as GA and ABA [80,81,82]. A similar situation may apply to other hormones as well [83,84,85,86]. It was reported that interactions among JAZ, ARF, and ABSCISIC ACID INSENSITIVE5 (ABI5) contribute to the crosstalk of JA, auxin, and ABA during the germination of Arabidopsis seeds [87]. ARFs and ABI5 are the core transcription factors of auxin and the ABA signaling pathway, respectively. ABI5 accumulation was reported to inhibit seed germination and early seedling establishment [88,89,90]. ARF 10 and ARF16 were reported to physically interact with ABI5, which activates the transcriptional function of ABI5 [87]. JAZ protein, the main inhibitor of the JA signaling pathway, competitively inhibits the interaction of ARF10/ARF16-ABI5 and thereby impairs ABI5 transcription [87,91]. Therefore, JA-mediated regulation of ABA signaling during seed germination may be synergistically regulated by auxin. We noticed that the expression of a gene encoding ABI5 was significantly down-regulated in germinating seeds (Figure 7). Although the specific regulatory mechanism needs further study, auxin and JA signaling may cooperate to regulate the expression of ABI5 during the germination of S. torvum seeds.
Seed dormancy-related genes were also investigated in this study (Table 1). DELAY OF GERMINATION1 (DOG1) gene is a master genetic regulator in determining the depth of seed dormancy [92]. Their amount is closely correlated with the dormancy level of freshly harvested seeds [93], and depression of DOG1 genes usually promotes seed germination [94]. In this study, we identified several DOG-coding genes that may be involved in the dormancy release and germination of S. torvum seeds (Table 1). Their expression was significantly down-regulated in germinated seeds but not in ungerminated seeds. This result suggests that the dormancy of GA-treated ungerminated seeds was not broken. We propose that the expression of DOG1-like genes may be employed as a dormancy indicator of S. torvum seeds. The regulatory role of DOG1 is considered to echo with ABA signaling, which affects the expression of ABI5 to regulate seed germination [95]. Given that the expression pattern of ABI5 was similar to that of DOG1 genes (Figure 6, Table 1), ABI5 may be a downstream target of DOG1 to regulate the germination of S. torvum seeds.
Other key regulators in seed dormancy and germination, such as Dormancy-associated DRM1-like protein, FUS3, LEC1, SOMNUS, and PIF1, showed a similar expression pattern with that of DOG1 (Table 1). DRM1 was found to be highly expressed in dormant seeds, while its ectopic expression delayed the germination of Arabidopsis seeds [96]. Previous studies reported that the expression of DOG1 relies directly or indirectly on FUS3 and LEC1 [97,98,99], which may involve SOMNUS and ABI3 in coordination with ABA signaling [95,100]. PIF1 interacts with REVEILLE 1 (RVE1) and they act as negative regulators in light-mediated seed germination via depression of the expression of GA3ox and thus the biosynthesis of active GA [101]. Thus, the down-regulation of these genes (Table 1) may contribute to the up-regulation of GA3ox (Figure 5) in germinated S. torvum seeds.

5. Conclusions

Exogenous application of 1.5 mM GA could effectively promote the germination of S. torvum seeds. GA treatment induces comprehensive changes in the expression of genes involved in energy metabolism, hormone biosynthesis, and signaling transduction. This study identified a large number of potential genes that are responsible for S. torvum seed germination, which provides theoretical basis for further research on the promotion of seed germination by GA.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14081736/s1: Figure S1: Germination percentage of S. torvum seeds under different treatments; Figure S2: Seed samples collected for RNA-seq analysis; Figure S3: Transcriptome analysis of germinated and ungerminated S. torvum seeds; Figure S4: KEGG pathways enriched in DEGs identified in each comparison group; Figure S5: Top 20 GO terms enriched in the DEGs in G group compared to W group; Figure S6: Top 20 GO terms enriched in the DEGs in G group compared to NG group; Figure S7: Top 20 GO terms enriched in the DEGs in NG group compared to W group; Figure S8: Overview of plant hormone signaling transduction pathway via KEGG analysis; Table S1: Statistics of S. torvum seed germination induced by 1.5 mM GA; Table S2: Statistics of RNA-seq read filter and mapping; Table S3: Raw data of gene expression; Table S4: List of novel genes identified by RNA-seq; Table S5: Statistics of PCA on RNA-seq data; Table S6: List of differentially expressed genes (DEGs); Table S7: List of potential 9723 genes induced by GA and responsible for germination; Table S8: List of GRGs involved in energy metabolism; Table S9: List of GRGs involved in hormone biosynthesis and signaling transduction; Table S10: List of transcription factors responsible for GA-induced germination of S. torvum.

Author Contributions

Conceptualization, L.T. and Y.Y.; methodology and validation, L.T. and Y.Y.; formal analysis, S.W., Y.Y. and L.T.; investigation, Q.S., K.Y., W.Z. and Y.Y.; data curation, L.Z.; writing—original draft preparation, S.W., Y.Y. and L.T.; writing—review and editing, L.Z. and T.W.O.; supervision, L.T.; project administration, L.T.; funding acquisition, L.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the “Pioneer” and “Leading Goose” R&D Program of Zhejiang (Grant No 2022C02051); the Zhejiang A&F University Starting Funds of Scientific Research and Development, Grant Nos 203402000101, 203402000501 and 203402001301; and the Student Scientific research training program of Zhejiang A&F University (Grant No S202210341138).

Data Availability Statement

The original data presented in the study are openly available in the the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/sra, accessed on 22 July 2024) with reference number PRJNA1138451.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Germination of S. torvum seeds treated by exogenous application of 1.5 mM GA. (A) Cumulative germination percentage (CGP) of S. torvum seeds. (B) Mean daily germination rate (MDG) of S. torvum seeds. (C) Germination Rate Index (GRI) of S. torvum seeds. X axis, days after incubation with 1.5 mM GA. The data were calculated based on five independent experiments.
Figure 1. Germination of S. torvum seeds treated by exogenous application of 1.5 mM GA. (A) Cumulative germination percentage (CGP) of S. torvum seeds. (B) Mean daily germination rate (MDG) of S. torvum seeds. (C) Germination Rate Index (GRI) of S. torvum seeds. X axis, days after incubation with 1.5 mM GA. The data were calculated based on five independent experiments.
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Figure 2. Analysis of differential expressed genes (DEGs) in germinated seeds in comparison to ungerminated seeds. (A) Number of DEGs identified among germinated (G) and ungerminated seeds (NG) treated by GA compared to control seeds treated by ddH2O. Blue, orange and gray bars represent the number of total, up-regulated, and down-regulated DEGs. (B) Heatmap analysis to show expression pattern of DEGs in each sample. (C) KEGG pathways enriched in DEGs identified in each comparison group.
Figure 2. Analysis of differential expressed genes (DEGs) in germinated seeds in comparison to ungerminated seeds. (A) Number of DEGs identified among germinated (G) and ungerminated seeds (NG) treated by GA compared to control seeds treated by ddH2O. Blue, orange and gray bars represent the number of total, up-regulated, and down-regulated DEGs. (B) Heatmap analysis to show expression pattern of DEGs in each sample. (C) KEGG pathways enriched in DEGs identified in each comparison group.
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Figure 3. Analysis of germination-related genes (GRGs). (A) Venn diagram analysis showing the overlap of DEGs among different comparison groups. (B) Flow chart to identify 9723 germination-related genes (GRGs). (C,D) KEGG pathway (C) and GO term (D) enrichment analyses of GRGs.
Figure 3. Analysis of germination-related genes (GRGs). (A) Venn diagram analysis showing the overlap of DEGs among different comparison groups. (B) Flow chart to identify 9723 germination-related genes (GRGs). (C,D) KEGG pathway (C) and GO term (D) enrichment analyses of GRGs.
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Figure 4. Expression of the annotated GRGs encoding the key enzymes related to energy metabolism. The pathways include amino sugar and nucleotide sugar metabolism, glycolysis, citrate cycle (TCA cycle), and pyruvate metabolism. The key compounds and substrates are shown in blue color. GlcNAc, N-Acetylglucosamine; GlcN, glucosamine; GlcNAc-6P, N-acetyl-D-glucosamine 6-phosphate; Fru-6-P, Fructose 6-phosphate; Glc-6-P, Glucose 6-phosphate; Glc-1P, Glucose-1-phosphate; UDP-Glc, UDP-glucose; UDP-GalA, UDP-α-d-galacturonic acid; Fru-1,6-P2, Fructose 1,6-bisphosphate; Glyceraldehyde-3P, Glyceraldehyde 3-phosphate; Glycerate-1,3P2, glycerate-1,3-bisphosphate; Glycerate-3P, glycerate 3-bisphosphate; Glycerate-2P, glycerate 2-bisphosphate; CHIT, endochitinase; HXK, hexokinase; UGE, UDP-glucose 4-epimerase; GAE, UDP-glucuronate 4-epimerase; GAUT, galacturonosyltransferase; G6PI, UTP-glucose-1-phosphate uridylyltransferase; PFK, ATP-dependent 6-phosphofructokinase; ALDA, fructose-bisphosphate aldolase; PGK, phosphoglycerate kinase; BDPM, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase; ENO1, enolase; PK, pyruvate kinase; ME, NAD-dependent malic enzyme; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ACO1, aconitate hydratase; IDH, isocitrate dehydrogenase; ACYL, ATP-citrate synthase; OGDH, 2-oxoglutarate dehydrogenase; DLD, dihydrolipoyl dehydrogenase; SDH, succinate dehydrogenase; FH, Fumarate hydratase; MDH, malate dehydrogenase.
Figure 4. Expression of the annotated GRGs encoding the key enzymes related to energy metabolism. The pathways include amino sugar and nucleotide sugar metabolism, glycolysis, citrate cycle (TCA cycle), and pyruvate metabolism. The key compounds and substrates are shown in blue color. GlcNAc, N-Acetylglucosamine; GlcN, glucosamine; GlcNAc-6P, N-acetyl-D-glucosamine 6-phosphate; Fru-6-P, Fructose 6-phosphate; Glc-6-P, Glucose 6-phosphate; Glc-1P, Glucose-1-phosphate; UDP-Glc, UDP-glucose; UDP-GalA, UDP-α-d-galacturonic acid; Fru-1,6-P2, Fructose 1,6-bisphosphate; Glyceraldehyde-3P, Glyceraldehyde 3-phosphate; Glycerate-1,3P2, glycerate-1,3-bisphosphate; Glycerate-3P, glycerate 3-bisphosphate; Glycerate-2P, glycerate 2-bisphosphate; CHIT, endochitinase; HXK, hexokinase; UGE, UDP-glucose 4-epimerase; GAE, UDP-glucuronate 4-epimerase; GAUT, galacturonosyltransferase; G6PI, UTP-glucose-1-phosphate uridylyltransferase; PFK, ATP-dependent 6-phosphofructokinase; ALDA, fructose-bisphosphate aldolase; PGK, phosphoglycerate kinase; BDPM, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase; ENO1, enolase; PK, pyruvate kinase; ME, NAD-dependent malic enzyme; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ACO1, aconitate hydratase; IDH, isocitrate dehydrogenase; ACYL, ATP-citrate synthase; OGDH, 2-oxoglutarate dehydrogenase; DLD, dihydrolipoyl dehydrogenase; SDH, succinate dehydrogenase; FH, Fumarate hydratase; MDH, malate dehydrogenase.
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Figure 5. Expression of GRGs encoding the key components related to GA biosynthetic and signaling pathways. (A) Diagram of the GA biosynthetic and signaling pathway. The key compounds and substrates in the GA biosynthetic pathway are shown in blue color. (B) Heatmap depicting the expression patterns of GRGs involved in GA biosynthesis and signaling. GGPP, trans-geranyl-geranyl diphosphate; ent-CDP, ent-copalyl diphosphate; CPS, copalyl diphosphate synthase; KS, ent-kaurene synthase; KO, ent-kaurene oxidase; KAO, ent-kaurenoic acid oxidase; GA13ox, gibberellin 13-oxidase; GA2ox, gibberellin 2-oxidase; GA20ox, gibberellin 20-oxidase; GA3ox, gibberellin 3-oxidase; GID1, gibberellin INSENSITIVE DWARF1; TF, transcription factor.
Figure 5. Expression of GRGs encoding the key components related to GA biosynthetic and signaling pathways. (A) Diagram of the GA biosynthetic and signaling pathway. The key compounds and substrates in the GA biosynthetic pathway are shown in blue color. (B) Heatmap depicting the expression patterns of GRGs involved in GA biosynthesis and signaling. GGPP, trans-geranyl-geranyl diphosphate; ent-CDP, ent-copalyl diphosphate; CPS, copalyl diphosphate synthase; KS, ent-kaurene synthase; KO, ent-kaurene oxidase; KAO, ent-kaurenoic acid oxidase; GA13ox, gibberellin 13-oxidase; GA2ox, gibberellin 2-oxidase; GA20ox, gibberellin 20-oxidase; GA3ox, gibberellin 3-oxidase; GID1, gibberellin INSENSITIVE DWARF1; TF, transcription factor.
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Figure 6. Expression of GRGs encoding key components related to abscisic acid (ABA) biosynthetic and signaling pathways. (A) Diagram of ABA biosynthetic and signaling pathway. The key compounds and substrates in ABA biosynthetic pathway are shown in blue color. BCH, beta-carotene hydroxylase; ZEP, zeaxanthin epoxidase; NSY, neoxanthin synthase; NCED, 9-cis-epoxycarotenoid dioxygenase; AS-SDR, ABA-specific short-chain dehydrogenase/reductase; AAO, ABA-specific aldehyde oxidase. (B) Heatmap depicting the expression patterns of GRGs involved in ABA biosynthesis and signaling. TRAB1, Transcription factor responsible for ABA regulation 1; ABI5, ABSCISIC ACID INSENSITIVE5.
Figure 6. Expression of GRGs encoding key components related to abscisic acid (ABA) biosynthetic and signaling pathways. (A) Diagram of ABA biosynthetic and signaling pathway. The key compounds and substrates in ABA biosynthetic pathway are shown in blue color. BCH, beta-carotene hydroxylase; ZEP, zeaxanthin epoxidase; NSY, neoxanthin synthase; NCED, 9-cis-epoxycarotenoid dioxygenase; AS-SDR, ABA-specific short-chain dehydrogenase/reductase; AAO, ABA-specific aldehyde oxidase. (B) Heatmap depicting the expression patterns of GRGs involved in ABA biosynthesis and signaling. TRAB1, Transcription factor responsible for ABA regulation 1; ABI5, ABSCISIC ACID INSENSITIVE5.
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Figure 7. Expression of GRGs encoding the key components related to auxin biosynthetic and signaling pathways. (A) Diagram of auxin biosynthetic and signaling pathway. The key compounds and substrates in auxin biosynthetic pathway are shown in blue color. ANS, anthranilate synthase; AnPRT, anthranilate phosphoribosyltransferase; TSB, Trp synthase α; TSB, Trp synthase β; TAA, L-tryptophan–pyruvate aminotransferase; TDC, tyrosine decarboxylase; AMI, amidase; YUCCA, flavin-containing monooxygenase; ALDH, aldehyde dehydrogenase; Aux1/LAX, AUXIN1/LIKE-AUX1; TIR1, TRANSPORT INHIBITOR RESPONSE 1; AUX/IAA, AUXIN/INDOLE ACETIC ACID; ARF, Auxin response factor; GH3, GRETCHEN HAGEN 3; SAUR, small auxin up-regulated RNA. (B,C) Heatmaps showing the expression patterns of GRGs involved in auxin biosynthesis and signaling, respectively. PIN, PIN-FORMED; ABP, auxin-binding protein; ILR1, IAA-amino acid hydrolase.
Figure 7. Expression of GRGs encoding the key components related to auxin biosynthetic and signaling pathways. (A) Diagram of auxin biosynthetic and signaling pathway. The key compounds and substrates in auxin biosynthetic pathway are shown in blue color. ANS, anthranilate synthase; AnPRT, anthranilate phosphoribosyltransferase; TSB, Trp synthase α; TSB, Trp synthase β; TAA, L-tryptophan–pyruvate aminotransferase; TDC, tyrosine decarboxylase; AMI, amidase; YUCCA, flavin-containing monooxygenase; ALDH, aldehyde dehydrogenase; Aux1/LAX, AUXIN1/LIKE-AUX1; TIR1, TRANSPORT INHIBITOR RESPONSE 1; AUX/IAA, AUXIN/INDOLE ACETIC ACID; ARF, Auxin response factor; GH3, GRETCHEN HAGEN 3; SAUR, small auxin up-regulated RNA. (B,C) Heatmaps showing the expression patterns of GRGs involved in auxin biosynthesis and signaling, respectively. PIN, PIN-FORMED; ABP, auxin-binding protein; ILR1, IAA-amino acid hydrolase.
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Figure 8. Expression of GRGs encoding the key components related to ethylene biosynthetic and signaling pathways. (A) Diagram of ethylene biosynthetic and signaling pathway. The key compounds and substrates in ethylene biosynthetic pathway are shown in blue color. SAMS, S-adenosylmethionine synthase; ACS, 1-aminocyclopropane-1-carboxylate synthase; ACO, 1-aminocyclopropane-1-carboxylate oxidase; ETR, ethylene receptor; CTR1, constitutive triple response 1; EIN2, ethylene-insensitive 2; EIN3, ethylene-insensitive 3; ERF, ethylene response factor. (B) Heatmap showing the expression patterns of GRGs involved in ethylene biosynthesis and signaling. (C) Heatmap depicting the expression patterns of the identified AP2/ERFs.
Figure 8. Expression of GRGs encoding the key components related to ethylene biosynthetic and signaling pathways. (A) Diagram of ethylene biosynthetic and signaling pathway. The key compounds and substrates in ethylene biosynthetic pathway are shown in blue color. SAMS, S-adenosylmethionine synthase; ACS, 1-aminocyclopropane-1-carboxylate synthase; ACO, 1-aminocyclopropane-1-carboxylate oxidase; ETR, ethylene receptor; CTR1, constitutive triple response 1; EIN2, ethylene-insensitive 2; EIN3, ethylene-insensitive 3; ERF, ethylene response factor. (B) Heatmap showing the expression patterns of GRGs involved in ethylene biosynthesis and signaling. (C) Heatmap depicting the expression patterns of the identified AP2/ERFs.
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Figure 9. Numbers of transcription factors (TFs) from the main TF families identified during germination of S. torvum seeds. (A) Pie chart showing the distribution of TFs from different families. (B,C) Expression pattern (left panel) and gene numbers (right panel) of down- (B) and up-regulated (C) TFs in germinated seeds. Gray lines in left panels represent the normalized expression level of each gene, while black thick lines represent the median centroids.
Figure 9. Numbers of transcription factors (TFs) from the main TF families identified during germination of S. torvum seeds. (A) Pie chart showing the distribution of TFs from different families. (B,C) Expression pattern (left panel) and gene numbers (right panel) of down- (B) and up-regulated (C) TFs in germinated seeds. Gray lines in left panels represent the normalized expression level of each gene, while black thick lines represent the median centroids.
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Table 1. Expression of known genes related to seed dormancy and germination.
Table 1. Expression of known genes related to seed dormancy and germination.
Gene IDGene DescriptionExpression Changes
G vs. WNG vs. WG vs. NG
Sol0222940.1DOG1-like protein−1.23−0.20−1.04
Sol0034020.1DOG1-like protein−6.52−0.40−6.13
Sol0108110.1DOG1-like protein−4.440.00−4.46
Sol0108120.1DOG1-like protein−4.51−0.51−4.02
Sol0108130.1DOG1-like protein−3.87−0.43−3.45
Sol0256750.1Dormancy-associated DRM1/APR-like protein−8.570.16−8.74
Sol0077770.1Dormancy-associated DRM1/APR-like protein−1.040.64−1.70
Sol0107130.1Dormancy-associated DRM1/APR-like protein−5.780.12−5.91
novel.4961FUSCA−2.88−0.27−2.63
Sol0168870.1LEAFY COTYLEDON 1-like−1.430.42−1.87
Sol0036660.1SOMNUS(SOM)−4.270.28−4.57
Sol0004150.1chromatin-remodeling factor PICKLE (PKL)-related protein−1.68−0.21−1.49
Sol0041910.1PIF4 like−1.96−0.19−1.79
novel.1047PIF1 like−4.85−0.06−4.81
novel.3095PIF1 like−4.45−0.67−3.80
novel.2961PIF1 like−2.440.05−2.51
novel.4424PIF1 like−1.850.19−2.06
novel.2810PIF1 like−1.28−0.26−1.03
Sol0116640.1REVEILLE 1 (RVE1) like−2.810.07−2.90
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Wu, S.; Si, Q.; Yang, K.; Zhang, W.; Zhang, L.; Okita, T.W.; Yan, Y.; Tian, L. Transcriptome Analysis Reveals the Effects of Exogenous Gibberellin on the Germination of Solanum torvum Seeds. Agronomy 2024, 14, 1736. https://doi.org/10.3390/agronomy14081736

AMA Style

Wu S, Si Q, Yang K, Zhang W, Zhang L, Okita TW, Yan Y, Tian L. Transcriptome Analysis Reveals the Effects of Exogenous Gibberellin on the Germination of Solanum torvum Seeds. Agronomy. 2024; 14(8):1736. https://doi.org/10.3390/agronomy14081736

Chicago/Turabian Style

Wu, Saimei, Qidong Si, Kejie Yang, Wenwei Zhang, Laining Zhang, Thomas W. Okita, Yanyan Yan, and Li Tian. 2024. "Transcriptome Analysis Reveals the Effects of Exogenous Gibberellin on the Germination of Solanum torvum Seeds" Agronomy 14, no. 8: 1736. https://doi.org/10.3390/agronomy14081736

APA Style

Wu, S., Si, Q., Yang, K., Zhang, W., Zhang, L., Okita, T. W., Yan, Y., & Tian, L. (2024). Transcriptome Analysis Reveals the Effects of Exogenous Gibberellin on the Germination of Solanum torvum Seeds. Agronomy, 14(8), 1736. https://doi.org/10.3390/agronomy14081736

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