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Article

Comparative Transcriptome Analysis Reveals the Mechanisms Underlying Differential Seed Vigor in Two Contrasting Peanut Genotypes

by
Shengyu Li
1,
Jiali Zeng
1,
Zhao Zheng
1,2,
Qi Zhou
1,
Shaona Chen
1,
Yixiong Zheng
1,
Xiaorong Wan
1 and
Bin Yang
1,*
1
Guangzhou Key Laboratory for Research and Development of Crop Germplasm Resources, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
2
School of Life Sciences, South China Normal University, Guangzhou 510631, China
*
Author to whom correspondence should be addressed.
Agriculture 2022, 12(9), 1355; https://doi.org/10.3390/agriculture12091355
Submission received: 26 July 2022 / Revised: 25 August 2022 / Accepted: 30 August 2022 / Published: 1 September 2022
(This article belongs to the Special Issue Genetics, Genomics and Bioengineering of Improved Legume Crops)
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Abstract

:
Seed vigor is an important agronomic trait, and wide variation exists among peanut accessions. However, the detailed regulatory mechanisms underlying differences in seed vigor between varieties are not known in peanut yet. Here, we performed a comparative transcriptome analysis of germinating seeds in two contrasting peanut accessions, namely A86 (high-vigor variety) and A279 (low-vigor variety). A total of 583 and 860 differentially expressed genes (DEGs) were identified at two imbibition stages between A86 and A279, respectively. Pathway enrichment tests highlighted the cell wall remodeling-, hormone signaling-, transcriptional regulation-, and oxidative stress-related DEGs, which may explain to a certain extent the difference in seed vigor between the two cultivars. Among them, the largest number of cell wall remodeling-related DEGs were extensions followed by cellulose synthases, fasciclin-like arabinogalactan proteins, polygalacturonases, expansins, and pectinesterases and the hormone signaling-related DEGs belonged mainly to the auxin and ethylene signaling pathway. The majority of transcriptional regulation-related DEGs were MYB, FAR1, and bHLH transcription factors, and the oxidative stress-related DEGs were mainly peroxidases. Further physiological analyses indicated that differences in seed vigor between A86 and A279 may be associated with differences in the ROS-scavenging abilities mediated by peroxidases. Moreover, we identified 16 DEGs homologous to known Arabidopsis regulators of seed dormancy and germination, suggesting that these DEGs would play similar functional roles during peanut seed germination. Our results not only provide important insights into the difference in seed vigor between varieties, but offer candidate genes that are worth investigating in future studies.

1. Introduction

Seed vigor is regarded as a critical agronomic trait related to uniform germination and seedling growth [1]. In general, seeds with high vigor germinate quickly and uniformly, showing more tolerance of various environmental stresses in the field than those with low vigor [2]. Peanut (Arachis hypogaea L.) is an economically important grain legume and oilseed crop that furnishes protein, edible vegetable oil, vitamins, and other micronutrients for humans [3,4]. Rapid and uniform seed germination is crucial for the establishment of strong seedlings and achieving high yield and quality in peanut. Therefore, it is critical to understand the molecular mechanisms of seed vigor for peanut breeding and field production.
Seed germination is a complex process commenced with the uptake of water by the metabolically quiescent dry seed and terminated with the protrusion of the embryonic axis or radicle [5]. The control of germination is considered to result from a counterbalance between the ability of the embryo to grow and the physical restrictions imposed by the surrounding endosperm and testa layers [6]. The cell wall plays a critical role in regulating the shape and integrity of the cell and provides mechanical strength to the high turgor pressure inside each cell [7]. Weakening of the micropylar endosperm requires cell wall loosening by wall hydrolases and finally leads to endosperm rupture and radicle emergence during seed imbibition, which is a widely conserved mechanism [8,9]. Cell wall integrity may not only affect seed germination directly through the physical properties of the seed cell wall but also indirectly through the modulation of various hormone signaling [10,11]. However, the functions of the cell wall in peanut remain largely unknown during seed germination.
The phytohormones abscisic acid (ABA) and gibberellin (GA) are the predominant regulators that antagonistically regulate seed dormancy and germination [12]. ABA is involved in initiating and maintaining seed dormancy, whereas GA contributes to breaking seed dormancy and promotes subsequent germination [13]. In addition to ABA and GA, other hormones including ethylene (ETH), brassinosteroid (BR), auxin (IAA), and cytokinin (CK) have also been reported to promote or inhibit seed germination [14,15,16,17,18]. The relationships between ABA/GA and other hormones in seed germination, however, are unclear. Transcription factors (TFs) play key roles in plant development through interaction with cis-regulatory elements of their target genes and/or other TFs [19]. Several key TFs have been identified as crucial components of the hormone signaling pathway and play essential roles in seed dormancy and germination [20,21,22,23]. Although transcriptional regulations are thought to be integrated into phytohormone signaling pathways for the control of seed germination, the precise mechanisms of this coupling are not yet well-understood in peanut.
Oxidative damage, mainly deriving from the excessive production of toxic reactive oxygen species (ROS), is the most common insult to cellular homeostasis in seed physiology [24]. Therefore, the tight regulation of steady-state levels of ROS seems to be necessary to prevent oxidative damage at subcellular levels, while simultaneously allowing ROS to perform important functions as signaling molecules in regulating seed germination [25]. Plant cells possess a complex antioxidant defense system for maintaining the balance of generated ROS, which can be classified into non-enzymatic antioxidants such as ascorbic acid and glutathione and enzymatic antioxidants such as catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) [26,27]. In the early stages of seed germination, moderate levels of ROS can act as important mediators in cell wall loosening and endosperm weakening [28]. Furthermore, the complex networks of cross-communication between ROS and the different hormone signaling pathways such as IAA, ABA, and ETH signaling have been shown to regulate plant growth and development [29,30]. In peanut, however, the key regulatory mechanism of ROS-mediated seed germination remains unclear.
In this study, we investigated the molecular mechanism of peanut seed germination by comparing the transcriptomes of high- and low-vigor cultivars at two different time points during seed imbibition. Transcriptome analysis highlighted several major molecular differences between the two cultivars, which may partly explain their differential seed vigor during seed germination. Our results provide new information regarding the molecular basis of peanut seed germination, which will facilitate the improvement of seed vigor in peanut breeding programs.

2. Materials and Methods

2.1. Plant Materials

In the preliminary experiment, the root phenotype of twenty advanced breeding lines of peanut was first observed and evaluated at 7 days after imbibition (Figure S1). The biggest difference in root length was found between A86 and A279 (Figure S1). However, there was no significant difference in grain length, grain width, and 100-grain weight between A86 and A279 (Figure S2). Finally, A86 and A279 were selected as the experimental material and planted in the field at the Huadu Experimental Station of Zhongkai University of Agriculture and Engineering (Guangdong Province, China). Peanut seeds were harvested in their maturity stage, dried for two weeks in the sun, and then used for the germination assay.

2.2. Evaluation of Seed Vigor

For each replicate, good quality uniform-sized 25 seeds from each variety were placed on the seed germination box with filter paper and 40 mL of distilled water. All seed germination boxes were incubated in a constant temperature (28 °C) incubator with a 12 h light/12 h dark cycle for 7 days. A total of six traits related to seed vigor including root length, root surface area, root volume, hypocotyl length, hypocotyl surface area, and hypocotyl volume were measured daily using a ScanMaker i800 Plus scanner (Microtek, Shanghai, China) in combination with an LA-S plant image analysis system (Hangzhou WSeen Detection Technology Co. Ltd., Hangzhou, China). Three biological replicates of each line were included in this study. The whole seeds at 2, 3, and 4 days after imbibition of each variety were snap-frozen in liquid nitrogen and stored in a refrigerator at −80 °C until use.

2.3. RNA Extraction, Transcriptome Sequencing, and Analysis

The total RNA was isolated using the Universal Plant RNA Extraction Kit (Bioteke, Beijing, China) according to the manufacturer’s instructions. The quality of the total RNA was confirmed using a Nanodrop 2000 spectrophotometry (Thermo Fisher Scientific, Wilmington, DE, USA) and agarose gel electrophoresis. Library construction and sequencing were performed on a BGISEQ platform by Beijing Genomic Institution (BGI, Shenzhen, China). The raw sequencing reads were cleaned to identify clean reads by removing low-quality reads and reads containing the adapter and unknown nucleotides. After read filtering, clean reads were mapped to the peanut reference genome (Tifrunner) using the HISAT/Bowtie2 tool [31,32,33]. The gene expression level was quantified as transcripts per million (TPM) reads using RSEM software [34]. Differentially expressed genes (DEGs) between the two varieties were determined using DESeq2 with a cutoff of a log2 fold change (log2FC) |log2FC| ≥ 1 and an adjusted p-value < 0.05 [35]. All the DEGs were further subjected to enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms.

2.4. Evaluation of Hydrogen Peroxide Level and Peroxidase Activity

The hydrogen peroxide (H2O2) content was determined based on the titanium-peroxide complex method [36]. Approximately 1 g of each sample was homogenized with 10 mL of phosphatic buffer solution (PBS) and centrifuged at 8000× g for 10 min. The reactive mixture, containing 25 μL of 50 mg/mL Ti(SO4)2, 25 μL of ammonia solution, and 250 μL supernatant, was centrifuged at 4000× g for 10 min. The precipitate was then solubilized with 250 μL of 2 M H2SO4 and the absorbance was measured at 415 nm. The POD activity was determined based on the guaiacol oxidation method [37]. Approximately 1 g of each sample was homogenized with 10 mL of 20 mM KH2PO4 and centrifuged at 8000× g for 10 min. The reactive mixture contained 120 μL of PBS, 30 μL of guaiacol, 30 μL of 30% H2O2, 60 μL distilled water, and 5 μL supernatant. The change in absorbance at 470 nm due to guaiacol oxidation was recorded. Quantitative analysis was performed using the predetermined calibration curves. Three biological replications were performed.

2.5. Gene Expression Analysis

Expression analyses were performed using quantitative real-time PCR (qRT-PCR). using SYBR qPCR Master Mix (Vazyme Biotech Co. Ltd., Nanjing, China) on a Bio-Rad CFX96 machine (Bio-Rad Laboratories Inc., Hercules, CA, USA). The peanut ACTIN gene was used as an internal standard to normalize the expression data in each sample. Relative expression values were calculated using the comparative Ct method [38]. The gene-specific primers used for qRT-PCR validation are listed in Table S1. Three biological replicates were performed.

2.6. Data Analysis

Experimental data were analyzed using SPSS Statistics (IBM, Chicago, IL, USA). The Student’s t-test was used to test for the significant differences at the 5% and 1% levels of probability.

3. Results

3.1. Differences in Seed Vigor between A86 and A279

To compare the characteristics of seed vigor between the peanut varieties A86 and A279, we measured the changes in the root and hypocotyl growth of imbibed seeds over the course of 7 days after imbibition. From the phenotypes shown in Figure 1, the root length, root surface area, root volume, hypocotyl length, hypocotyl surface area, and hypocotyl volume of A86 were significantly higher than that of A279 during seed germination and seedling growth. These results indicated that A86 exhibited higher seed vigor than A279.

3.2. Comparison of Transcriptome between A86 and A279

To identify transcriptional changes responsible for the difference in seed vigor between A86 and A279, RNA-Seq analysis was performed on the whole germinating seeds at 2 and 3 days after imbibition (hereafter referred to as A86-2d, A279-2d, A86-3d, and A279-3d, respectively). Three independent biological replicates for each time point of the selected seed tissues were used to construct 12 independent RNA-sequencing libraries. Ultimately, on average, 23.79 million high-quality reads per replicate of the samples were generated from the clean RNA-Seq data, 97.38% of which could be aligned to a reference genome of the cultivated peanut species Tifrunner [33]. The detailed statistics of the sequencing are shown in Table S2. Based on a significance threshold of |log2FC| ≥1 and adjusted p-value < 0.05, a total of 583 (338 upregulated and 245 downregulated) and 860 (623 upregulated and 237 downregulated) differentially expressed genes (DEGs) were identified in the A86-2d vs. A279-2d and A86-3d vs. A279-3d comparisons, respectively (Figure 2A). In total, 296 DEGs (145 upregulated and 151 downregulated) were commonly detected in both comparisons (Figure 2B). These results indicated that the peanut varieties A86 and A279 showed different dynamic transcription profiles during seed germination.

3.3. Functional Classification of DEGs

To explore the possible biological functions related to peanut seed germination, KEGG and GO analyses were further performed to classify the DEGs identified in the A86-2d vs. A279-2d and A86-3d vs. A279-3d comparisons. Figure 3A,B illustrates the main KEGG pathway of each comparison in the KEGG analysis. Among them, several KEGG pathways were shared in both comparisons including plant hormone signaling transduction, phenylpropanoid biosynthesis, carbon metabolism, biosynthesis of antibiotics, protein processing in endoplasmic reticulum, and glycolysis/gluconeogenesis. Likewise, Figure 3C,D illustrates the main GO terms of each comparison in the GO analysis. This approach unveiled several GO terms including defense response, regulation of transcription, hormone-mediated regulation, cell wall remodeling, and oxidative stress as being commonly identified in both comparisons. These data provided an initial framework for the functional classification related to seed germination in peanut.

3.4. DEGs Involved in Cell Wall Remodeling

More than 100 DEGs involved in cell wall remodeling were identified in the comparisons of A86-2d vs. A279-2d and A86-3d vs. A279-3d (Table S3). These DEGs were mainly classified into several groups including extensin (EXT), cellulose synthase (CESA), fasciclin-like arabinogalactan protein (FLA), polygalacturonase (PG), expansin (EXP), and pectinesterase (PE) (Figure 4A). The majority of these DEGs were expressed at higher levels in A86 than in A279, especially at the 3-day time point (Figure 4B). In contrast, only five DEGs were expressed at lower levels in A86 than in A279 during seed germination including two DEGs encoding PG, two DEGs encoding POD, and one DEG encoding EXP (Figure 4B). These results indicate that there were differences in th cell wall biosynthesis and degradation between A86 and A279 during seed germination.

3.5. DEGs Involved in Hormone Signaling Transduction

There were 32 DEGs involved in hormone signaling transduction in the comparisons of A86-2d vs. A279-2d and A86-3d vs. A279-3d (Table S4). In comparison, the DEGs linked to the IAA and ETH signaling pathways tended to be more numerous than those involved in other hormone signaling pathways (Figure 5A). In the IAA signaling pathway, 16 DEGs were identified, which were annotated as auxin response factor (ARF), Aux/IAA protein (Aux/IAA), indole-3-acetic acid-amido synthetase (GH3), and auxin-responsive protein (SAUR) (Figure 5B). Almost all of the DEGs related to IAA signaling were significantly upregulated in A86 compared to A279 across the two stages, except for one DEG encoded ARF, which was significantly downregulated (Figure 5B). In the ETH signaling pathway, seven DEGs were identified including one annotated as serine/threonine-protein kinase (CTR1) and five annotated as ethylene-responsive transcription factors (ERF), all of which were more highly expressed in A86 than in A279 throughout the two stages (Figure 5C). These results indicate that the IAA and ETH signaling transduction-related genes are more active in A86 than in A279 during seed germination.

3.6. DEGs Involved in Transcriptional Regulation

A total of 82 DEGs encoding TFs were identified in the comparisons of A86-2d vs. A279-2d and A86-3d vs. A279-3d (Table S5), which mainly consisted of the MYB, FAR1, bHLH, AP2, ZFP, AT-hook, and Homebox families (Figure 6A). Members of the MYB, FAR1, and bHLH families represented the largest number of differentially expressed TFs (Figure 6A). Among them, 13 upregulated genes and five downregulated genes were MYB domain-containing TFs; six upregulated genes and eight downregulated genes were characterized as FAR1 TFs; bHLH TFs included nine upregulated genes and two downregulated genes (Figure 6B). These results indicate that distinct patterns of transcriptional regulation occurred in A86 and A279 during seed germination.

3.7. DEGs Involved in Oxidative Stress

There were 29 DEGs related to oxidative stress in the comparisons of A86-2d vs. A279-2d and A86-3d vs. A279-3d (Table S6). Intriguingly, we identified 18 DEGs encoding peroxidase (POD), which accounted for the largest proportion of DEGs related to oxidative stress (Figure 7A). The majority of POD genes were more highly expressed in A86 than in A279, especially at the 3-day time point (Figure 7B). Meanwhile, the physiological analysis showed that the relative levels of hydrogen peroxide (H2O2) were significantly higher in A279 than in A86 (Figure 7C), while the higher activities of POD were observed in A86 compared to A279 at both the two-time points (Figure 7D). These results indicate that the ROS-scavenging ability of A86 was stronger than that of A279 during seed germination.

3.8. Analysis of DEGs Associated with Seed Germination Based on Known Arabidopsis Genes

To date, hundreds of genes have been reported to be involved in the regulation of seed germination in the model plant Arabidopsis according to the TAIR (The Arabidopsis Information Resource; https://www.arabidopsis.org/index.jsp; accessed on 6 March 2022) database. In this study, a BLASTP search of the Arabidopsis protein database allowed us to identify 16 DEGs possibly involved in the regulation of seed germination (Figure 8). For example, LOC112783336, LOC112697826, and LOC112784649 were identified as orthologues of Arabidopsis EXP2, PRX16, and PRX71, which have been shown to be involved in seed germination mediated by cell wall metabolism (Figure 8). LOC112710147, LOC112779231, LOC112742696, LOC112733034, LOC112791070, LOC112750025, LOC112801110, LOC112750697, LOC112715355, and LOC112797092 were identified as orthologues of Arabidopsis PELPK1, ICE1, PRT6, DOG1, PUB18, PUB19, and PYL4, which have been characterized to play important roles in hormone-mediated seed germination (Figure 8). In addition, it is worth mentioning that several expansins (EXPs), DOG1-like, ABA receptor (PYLs), and plant U-boxes (PUBs) genes, identified in rice, wheat, barley, soybean or oilseed rape, have also been demonstrated to regulate seed germination or dormancy [39,40,41,42,43,44,45,46]. These results indicate that these DEGs might serve similar functions during peanut seed germination.

3.9. Confirmation of the Differential Gene Expression

In order to validate the results of the RNA-Seq, three DEGs including LOC112776756 (encoding auxin-responsive protein IAA14; designated as AhIAA14), LOC112712392 (encoding extension; designated as AhEXT), and LOC112783336 (encoding expansin; designated as AhEXP) were selected randomly for the measurement of mRNA levels using the qRT-PCR method. As shown in Figure 9, the expression trends of these genes were generally consistent with those of the RNA-Seq analysis, indicating that the transcriptome results were credible in this study.

4. Discussion

Seed germination is an important agronomic trait in determining the seedling growth [47]. Currently, the molecular mechanisms underlying seed germination have been extensively studied in the model plant Arabidopsis, but not yet in peanut. Here, we performed a comparative transcriptome analysis between the high seed vigor variety A86 and low seed vigor variety A279 during seed imbibition, and the DEGs were identified and further analyzed, which could provide valuable insights into the candidate genes underlying seed vigor in peanut. To the best of our knowledge, this is the first report on the application of the transcriptomic approach to identify transcriptional changes of two contrasting peanut genotypes during seed germination.
Proper cell wall remodeling is essential for both embryo cell expansion and the weakening of embryo-enclosing tissues to the completion of seed germination [48]. Several reports have shown that cell wall-related genes are highly expressed throughout germination and post-germination [49,50]. In Setaria italica, a comparative transcriptome analysis of the two cultivars revealed that a range of gene families associated with cell wall modification plays a role in seed germination under salinity stress. For example, the overexpression of cell wall-related gene SiKOR1 in Arabidopsis significantly affected the seed germination under salt condition [51]. In this study, many DEGs associated with cell wall loosening, expansion, or strengthening such as extension, cellulose synthase, fasciclin-like arabinogalactan protein, pectinesterase, and expansin were significantly upregulated in the high seed vigor variety A86 compared to the low seed vigor variety A279. The function analysis of cell wall-related genes has been shown to regulate seed germination in Arabidopsis by modulating the cell wall properties. For example, the activation of the extension gene AtEPR1 might have an important role in modifying the cell wall structure during seed germination, thus facilitating the protrusion of the radicle through the seed coat [52]. Mutation of cellulose synthase AtCESA1 exhibited significant inhibitory effects on cellulose synthesis, seed germination, hypocotyls, and primary roots elongation [53]. AtEXP2 (homologous with LOC112783336; Figure 8), an expansin-encoding gene, has been reported to be involved in GA-mediated regulation of the seed germination process by weakening the cell wall in endosperm and other tissues surrounding the embryo [54]. In this study, we speculated that the activation of cell wall-related genes might contribute to the growth regulation at the embryonic axes (hypocotyl and radicle) and help facilitate quick seedling establishment in peanut, which likely explains the observed difference in seed vigor between A86 and A279. Therefore, further studies are needed to determine the biological functions of cell wall-related genes during peanut seed germination.
The central role of ABA and GA in seed dormancy and germination has been well-established. Apart from ABA and GA, additional hormones such as IAA and ETH also influence seed germination. IAA has been demonstrated to regulate seed germination both positively and negatively depending on its dose [55], while ETH can inhibit seed dormancy establishment and promote seed germination [56]. In this study, DEGs involved in the signaling pathways of multiple hormones were identified during seed germination, with some of the most prominent being those associated with IAA and ETH signaling. Recently, the molecular regulations of IAA and ETH signaling during seed germination have been characterized in Arabidopsis. Genetic analyses revealed that the Aux/IAA protein AtIAA8 positively regulated seed germination through the transcriptional suppression of ABA Insensitive 3 (ABI3) by inhibiting ARF proteins [18]. Ethylene response factors AtERF15, AtERF55, and AtERF58 have been reported to be involved in the ABA- or GA-mediated regulation of germination [57,58], while AtERF12 participated in the regulation of seed dormancy by binding to the promoter of DOG1 (homologous with LOC112733034 and LOC112791070; Figure 8) and suppressing its expression [59]. In rice, an APETALA2/ethylene responsive factor OsEBP89 was shown to be induced by submergence stress and inhibited by drought stress, and loss of OsEBP89 improved the submergence and drought stress tolerance during seed germination [60]. In this study, we speculated that the transcriptional difference of the IAA and ETH signaling pathways could account for the germination difference between A86 and A279. Therefore, the involvement of IAA and ETH signaling in regulating seed germination is undoubtedly worth further investigation in peanut.
Recent transcriptome analyses have implied that several TF families might be involved in driving seed germination [61,62]. In this study, the DEGs encoding various putative TFs including MYB, FAR1, and bHLH were highly represented in both comparisons. Several studies have reported that MYB, FAR1, and bHLH TFs may function in the hormone-mediated regulation of seed germination in Arabidopsis. AtMYB7 was identified to negatively regulate ABA-imposed inhibition of seed germination by lowering the expression of ABA Insensitive 5 (ABI5), which is a key component in ABA signaling involved in repressing seed germination [63]. FAR1 and its homolog FHY3 were demonstrated to activate the expression of ABI5 by directly binding to its promoter to regulate seed germination and seedling growth in response to ABA [64]. The TF ICE1 (homologous with LOC112779231; Figure 8), a member of the bHLH family, functioned as a negative regulator to fine-tune ABA signaling by interacting with ABI5 and DELLA proteins and binding to the promoter of ABI3, thereby regulating seed dormancy and germination [65,66]. Downregulation or upregulation of MYB TF AtRSM1 expression changed the sensitivity of seed germination to ABA, salt, and osmotic stresses [67]. However, whether the TFs identified in our study also function in the hormone-mediated regulation of seed germination is unknown in peanut and deserves further investigation.
At physiologically low levels, ROS can act as redox messengers in signaling transduction pathways that control several processes including seed germination, whereas excess ROS accumulation induces oxidative stress in cells [25,68]. In plants, PODs are evolutionarily conserved antioxidant enzymes encoded by a large multi-gene family known to keep ROS at steady-state levels to avoid cell damage [69]. A previous study has shown that PODs were specifically expressed in the endosperm cap and radicle of germinating seeds [70]. In rice, an ascorbate peroxidase OsAPX1 has recently been shown to regulate seed germination under stress condition and ABA application [71,72]. In this study, we found that oxidative stress-related genes, mainly PODs, were significantly upregulated in A86 compared to A279. Consistently, the significantly higher activities of PODs were observed in A86, leading to less H2O2 accumulation, which indicate that this difference in the ability of ROS-scavenging might account for the difference in germination and growth speed of the two varieties. PODs have also been shown to control cell wall loosening that is probably necessary for endosperm weakening and radicle emergence in seeds [73,74,75]. In this study, two POD genes (LOC112779231 and LOC112779231; Figure 8) were identified as orthologues of Arabidopsis AtPrx16 and AtPrx71, which have been shown to be co-expressed in the micropylar endosperm and control cell wall loosening or stiffening during seed germination [75]. Accordingly, here we speculated that PODs might play dual roles during peanut seed germination, the first in the general control of ROS homeostasis and the second in mediating cell wall remodeling, but these would need further investigation.

5. Conclusions

In this study, transcriptome sequencing was first performed to investigate the patterns of gene expression during seed germination in two contrasting peanut genotypes. To understand the regulatory mechanism of seed vigor in peanut, the DEGs between the two varieties during seed germination were analyzed, and 583 and 860 genes showed significantly different expression in the high seed vigor variety compared to the low seed vigor variety during the two imbibition stages, respectively. Functional classification of these DEGs revealed that various biological processes such as cell wall remodeling, hormone signaling, transcriptional regulation, and oxidative stress may be functionally associated with differences in the germination phenotype between the two cultivars. In addition, several DEGs were identified as candidate regulators in seed germination based on the known function of Arabidopsis orthologs. This study provides valuable information for future research into the molecular mechanism of peanut seed vigor.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture12091355/s1, Figure S1: The root length of twenty advanced lines of peanut at 7 days after imbibition. Figure S2: A comparison of the grain size and weight between A86 and A279. Table S1: The primer pairs used in this study; Table S2: A summary of the read mapping statistics; Table S3: The DEGs related to cell wall remodeling; Table S4: The DEGs related to hormone signal transduction; Table S5: The DEGs related to transcriptional regulation; Table S6: The DEGs related to oxidative stress.

Author Contributions

Conceptualization, B.Y. and X.W.; Investigation, S.L., J.Z., Z.Z., Q.Z. and S.C.; Writing—original draft preparation, S.L. and B.Y.; Writing—review and editing, B.Y. and X.W.; Visualization, S.L. and B.Y.; Supervision, B.Y., Y.Z. and X.W.; Project administration, B.Y., Y.Z. and X.W.; Funding acquisition, B.Y. and X.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant No. 32101802, 32071737 and 32111530289), the Department of Education of Guangdong Province (Grant No. 2020ZDZX1013), the Department of Science and Technology of Guangdong Province (Grant No. 2021A050530073), and the Agricultural and Rural Department of Guangdong Province (Grant No. KB1708008).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request to the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. A comparison of the seed vigor between A86 and A279. (A) Phenotypes of root growth and hypocotyl elongation of A86 and A279 from 2 to 7 days. (B) Changes in root length, root surface area, root volume, hypocotyl length, hypocotyl surface area, and hypocotyl volume of A86 and A279 from 2 to 7 days. For statistical analysis, three independent experiments were conducted, each with three replicates (25 seeds for each replicate). Differences between A86 and A279 at each time point were analyzed by the Student’s t-test. * and ** indicate significant differences at p < 0.05 and p < 0.01, respectively.
Figure 1. A comparison of the seed vigor between A86 and A279. (A) Phenotypes of root growth and hypocotyl elongation of A86 and A279 from 2 to 7 days. (B) Changes in root length, root surface area, root volume, hypocotyl length, hypocotyl surface area, and hypocotyl volume of A86 and A279 from 2 to 7 days. For statistical analysis, three independent experiments were conducted, each with three replicates (25 seeds for each replicate). Differences between A86 and A279 at each time point were analyzed by the Student’s t-test. * and ** indicate significant differences at p < 0.05 and p < 0.01, respectively.
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Figure 2. A summary of the DEGs. (A) The number of DEGs between A86 and A279 at 2 and 3 days after imbibition. Venn diagrams showing the overlap of the upregulated (B) and downregulated (C) DEGs, respectively.
Figure 2. A summary of the DEGs. (A) The number of DEGs between A86 and A279 at 2 and 3 days after imbibition. Venn diagrams showing the overlap of the upregulated (B) and downregulated (C) DEGs, respectively.
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Figure 3. The functional GO and KEGG pathway classification of the DEGs. (A,B) The main GO terms of the DEGs between A86 and A279 at each time point. (C,D) The main KEGG pathways of the DEGs between A86 and A279 at each time point.
Figure 3. The functional GO and KEGG pathway classification of the DEGs. (A,B) The main GO terms of the DEGs between A86 and A279 at each time point. (C,D) The main KEGG pathways of the DEGs between A86 and A279 at each time point.
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Figure 4. The expression of DEGs involved in cell wall remodeling. (A) Pie chart showing the frequency distribution of DEGs involved in cell wall remodeling. (B) Heat-map showing the differences in the expression of representative genes related to cell wall remodeling.
Figure 4. The expression of DEGs involved in cell wall remodeling. (A) Pie chart showing the frequency distribution of DEGs involved in cell wall remodeling. (B) Heat-map showing the differences in the expression of representative genes related to cell wall remodeling.
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Figure 5. The expression of DEGs involved in hormone signal transduction. (A) Pie chart showing the frequency distribution of DEGs involved in hormone signal transduction. (B,C) Heat-map showing the differences in the expression of representative genes related to the IAA and ETH signaling pathways.
Figure 5. The expression of DEGs involved in hormone signal transduction. (A) Pie chart showing the frequency distribution of DEGs involved in hormone signal transduction. (B,C) Heat-map showing the differences in the expression of representative genes related to the IAA and ETH signaling pathways.
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Figure 6. The expression of DEGs involved in transcriptional regulation. (A) Pie chart showing the frequency distribution of DEGs involved in transcriptional regulation. (B) Heat-map showing the differences in the expression of representative transcription factor families.
Figure 6. The expression of DEGs involved in transcriptional regulation. (A) Pie chart showing the frequency distribution of DEGs involved in transcriptional regulation. (B) Heat-map showing the differences in the expression of representative transcription factor families.
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Figure 7. The expression of DEGs involved in oxidative stress. (A) Pie chart showing the frequency distribution of DEGs involved in oxidative stress. (B) Heat-map showing the differences in the expression of representative genes related to the ROS signaling pathway. (C,D) Comparison of the relative H2O2 level and POD activity at different time points (2, 3, and 4 days after imbibition) between A86 and A279. Differences between A86 and A279 at each time point were analyzed by the Student’s t-test. ** indicates significant difference at p < 0.01.
Figure 7. The expression of DEGs involved in oxidative stress. (A) Pie chart showing the frequency distribution of DEGs involved in oxidative stress. (B) Heat-map showing the differences in the expression of representative genes related to the ROS signaling pathway. (C,D) Comparison of the relative H2O2 level and POD activity at different time points (2, 3, and 4 days after imbibition) between A86 and A279. Differences between A86 and A279 at each time point were analyzed by the Student’s t-test. ** indicates significant difference at p < 0.01.
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Figure 8. A heat-map showing the differences in expression of 16 genes possibly involved in seed germination.
Figure 8. A heat-map showing the differences in expression of 16 genes possibly involved in seed germination.
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Figure 9. The qRT-PCR validations. (A) AhIAA14. (B) AhEXT. (C) AhEXP. The relative expression levels of the three DEGs were calibrated against those of the Actin gene quantified by qRT-PCR.
Figure 9. The qRT-PCR validations. (A) AhIAA14. (B) AhEXT. (C) AhEXP. The relative expression levels of the three DEGs were calibrated against those of the Actin gene quantified by qRT-PCR.
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Li, S.; Zeng, J.; Zheng, Z.; Zhou, Q.; Chen, S.; Zheng, Y.; Wan, X.; Yang, B. Comparative Transcriptome Analysis Reveals the Mechanisms Underlying Differential Seed Vigor in Two Contrasting Peanut Genotypes. Agriculture 2022, 12, 1355. https://doi.org/10.3390/agriculture12091355

AMA Style

Li S, Zeng J, Zheng Z, Zhou Q, Chen S, Zheng Y, Wan X, Yang B. Comparative Transcriptome Analysis Reveals the Mechanisms Underlying Differential Seed Vigor in Two Contrasting Peanut Genotypes. Agriculture. 2022; 12(9):1355. https://doi.org/10.3390/agriculture12091355

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Li, Shengyu, Jiali Zeng, Zhao Zheng, Qi Zhou, Shaona Chen, Yixiong Zheng, Xiaorong Wan, and Bin Yang. 2022. "Comparative Transcriptome Analysis Reveals the Mechanisms Underlying Differential Seed Vigor in Two Contrasting Peanut Genotypes" Agriculture 12, no. 9: 1355. https://doi.org/10.3390/agriculture12091355

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