PEG-6000 Priming Improves Aged Soybean Seed Vigor via Carbon Metabolism, ROS Scavenging, Hormone Signaling, and Lignin Synthesis Regulation

: Seed priming, a valuable seed pretreatment method widely employed in agricultural production, counteracts the decline in seed vigor attributed to aging and deterioration. However, PEG priming effectively enhances the vigor of aged soybean seeds. In this study, “TONGDOU13” soybean seeds were subjected to PEG-6000 priming at varying concentrations (10%, 20%, 30%, 40%) for three different durations (12 h, 24 h, 36 h). The results showed that a 24 h priming with 30% PEG-6000 signiﬁcantly enhances the vigor of aged soybean seeds. To elucidate the mechanism underlying the heightened vigor resulting from PEG-6000 priming, we employed transcriptome sequencing and physiological–biochemical tests. Transcriptome sequencing analysis showed the signiﬁcant down-regulation of carbon metabolism-related genes post PEG-6000 priming, which facilitated energetically efﬁcient germination. Five peroxidase-encoding genes displayed signiﬁcant up-regulation, promoting the conversion of coumaryl alcohol to hydroxy-phenyl lignin, a probable catalyst for augmented seed vigor. SOD and GST genes were signiﬁcantly up-regulated, enhancing the scavenging ability of reactive oxygen species (ROS). The concurrent up-regulation of brassinolide (BR) and auxin (IAA) signals countered ABA signaling, thereby promoting aged seed germination. Further investigation included the measurements of antioxidant enzyme activity, hormone levels, and lignin content. Notably, primed aged seeds exhibited enhanced ROS scavenging ability, and increased lignin, BR, and IAA contents. Therefore, PEG priming may improve aged soybean seed vigor through the co-regulation of carbon metabolism, ROS scavenging, hormone signaling, and lignin synthesis. This study will be vital for preserving germplasm resources and reutilizing aged soybean seeds.


Introduction
Soybean is one of the most economically important legume crops globally, providing protein, oil, and other nutrients.As the world's population grows, so does the demand for soybeans.However, soybean production is largely affected by seed quality.Due to its high oil content, natural storage conditions can induce aging and deterioration, leading to the production of harmful substances.This, in turn, diminishes seed vigor, thereby impairing soybean yield potential [1][2][3].Seed aging and deterioration signify the inevitable transition from physiological maturity to senescence.This progression involves internal changes, including DNA damage [4], protein denaturation [5], the disruption of nucleic acid synthesis systems [1], the exacerbation of lipid peroxidation [6], and mitochondrial oxidative damage [7][8][9].External manifestations comprise sluggish and irregular seedling emergence, reduced germination rates, and weakened environmental stress resistance.Consequently, both the biological and economic yields of crops experience significant reductions [10][11][12][13].Nonetheless, this decline in vigor can be mitigated through seed priming techniques.
Seed priming, a method involving controlled water absorption followed by controlled dehydration before embryo breakthrough, allows the commencement of germinationrelated events but prevents radicle emergence.This technology enhances seed germination vigor by adjusting internal physiological activities and metabolism [14].According to current research, the methods include water priming, osmotic priming, hormone priming, chemical priming, etc. [15].These methods can effectively enhance seed vigor.Research demonstrates that NO priming enhances germination rates and antioxidant properties in deteriorated oat seeds, thus alleviating aging-related damages [16][17][18].Scholars have employed BR as a stimulant for priming tomato and rye seeds, thereby promoting germination, radical elongation, and hypocotyl growth [19,20].Additionally, melatonin priming has shown promise in improving soil salt tolerance and mitigating salt stress damage in cotton [21].
Among the factors affecting seed germination, the water availability is the most determinant factor as it participates in various metabolic activities and affects the growth of the embryonic axis.Previous studies have shown that environmental stresses may also positively influence seed performance as the internal regulation of seeds can improve seed vigor [22].One of methods for controlling the imbibition rate of the seeds uses osmotic solutions of PEG: it is inert, is not absorbed by the seed, but can control the slow water absorption.It enhances germination metrics in wheat seeds, including germination rate, vigor, and vigor index [23].In cotton seeds, PEG augments SOD and POD enzyme activity under salt stress, reduces MDA content, and increases salt tolerance [24].Additionally, PEG priming improves germination and seedling-stage photosynthesis while diminishing free ROS in rice seeds under drought stress [25].Aged sunflower seeds' germination was enhanced by PEG priming [26].Furthermore, aged oat seeds exhibited an increased vigor index after 12 h exposure to −1.2 MPa PEG priming [27].
Advancements in omics and molecular biology have elucidated molecular mechanisms behind seed germination, and induced plant resistance by osmopriming has been previously reported.CaCl 2 pretreatment reportedly triggered ROS release, enhancing rice resistance [28].PEG priming regulated α-extender gene expression in Arabidopsis seeds [29].Furthermore, PEG priming also modulates GR1, GR2, Amy2A, and Amy3A expression in rice seeds, thereby mitigating stress damage from nano-zinc oxide [30].PEG priming had a protective effect, maintaining the expression of HSP70 in rice seeds under normal and drought conditions [25].PEG priming induce the expression of BnPIPI, promoting water transportation required for the enzymatic metabolism of nutrient storage in early seed germination [31].In addition, PEG priming may enhance the salt tolerance of alfalfa seeds through memory H 2 O 2 signal transduction and the up-regulation of heme oxygenase [32].However, very few reports have elucidated the underlying molecular mechanism through which PEG enhances aged soybean seed vigor.This study aims to explore the impact of PEG priming on aged soybean seeds' vigor and investigate the morphological, physiological, biochemical, and differential gene expression changes post-priming.This will explain the molecular mechanism underlying PEG priming's efficacy in enhancing aged soybean seed vigor.

Materials, Seed Vigor Testing, Seed Aging, and Seed Priming Treatment
The seed "TONGDOU13" was chosen by the Jiangsu Yanjiang Institute of Agricultural Sciences, with new seeds being annually propagated.Seed vigor was determined via the ISTA germination method (http://www.seedtest.org,accessed on 7 November 2023).The new seeds were placed in an aging box with a 45 • C temperature and 100% humidity, treated with artificial accelerated aging for 24 h, and then dried at 25 • C to constant weight.After sterilization, the germination papers were soaked in deionized water.New seeds and aged seeds were disinfected with 75% alcohol for 15 s, washed with deionized water, placed on two layers of ger-mination paper, covered with another piece of germination paper (three biological replicates), rolled up, vertically placed in a self-sealing bag, subjected to a 25 • C and 8 h light/16 h dark treatment, and germinated for eight days.The standard germination rate (SG) and accelerated aging germination rate (AA) were detected, respectively.
Aged seeds were disinfected with 75% alcohol for 15 s and washed with deionized water.Priming treatments using 20% (PEG20P), 30% (PEG30P), and 40% (PEG40P) PEG-6000 concentration solutions were administered for 24 h with a 25 • C temperature.The optimal priming concentration was selected for 12 h, 24 h, and 36 h treatments with a 25 • C temperature, compared with hydro priming (HP) for aging seeds as the control.Following priming, seeds were washed with deionized water, dried at 25 • C to constant weight, placed on germination paper soaked with distilled water, and subjected to a 25 • C and 8 h light/16 h dark treatment.A paper roll germination test was performed.After 24 h of water absorption, samples were subsequently frozen in liquid nitrogen and finally stored at −80 • C for further use.

Morphology and Biomass Assays
The measurement was conducted according to the ISTA rules.After eight days of germination, the germination rate, germination index, and vigor index (50 seeds, three biological replicates) of each treatment were calculated, and five plants were randomly selected from each treatment for triplicates.The root length and seedling length were measured using a ruler, and the fresh weight and dry weight (dried at 55 • C to constant weight) of seedlings were measured using an electronic balance.GR(%) = Total number of normally germinated seeds/Number of seeds tested × 100 GI = ∑ Gt/Dt(Gt is the GR corresponding to Dt, and Dt is the day of the germination test) VI = GI × W(W is the fresh weight of germinated seeds).

Transcriptome Sequencing and Bioinformatics Analysis
After germinating and absorbing water for 24 h, the samples were sequenced from different seed groups: non-primed treated new seeds (CK0), non-primed treated aged seeds (CK1), aged seeds primed for 12 h (T1), and aged seeds primed for 24 h (T2) at 30% PEG-6000 concentration were sequenced for transcriptomes.Each treatment had triplicates.Total RNA (5 µg) was extracted from both primed and un-primed seeds using the TRIzol Kit (Invitrogen, Carlsbad, CA, USA).RNA concentration was measured using an ultra-micro spectrophotometer NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was assessed using the Agilent 2100 Bioanalyzer.The cDNA library was constructed using a TruSeq TM RNA kit and sequenced on an Illumina No-vaSeq6000 by Gene (Guangzhou, China).SOAPfuse software filtered the data to obtain clean data, and Tophat software aligned the filtered data with the soybean reference genome (https://phytozome-next.jgi.doe.gov/info/Gmax_Wm82_a2_v1,version Glyma2.0,accessed on 7 November 2023), allowing for two-base mismatches [33,34].After that, the differential expression multiple of this gene in different samples was calculated by using gene expression level FPKM as the unit.The differential expression gene (DEG) in samples was obtained using the criteria "FDR ≤ 0.05 and |log 2 Ratio (FC, Fold Change)| ≥ 1" The threshold for significance was "p-value ≤ 0.05".Blast2GO-v2.5 software was used for GO annotation analysis with "p-value ≤ 0.05".Finally, pathway enrichment analysis was conducted using Blast2GO-v2.2.26 software.

qRT-PCR Analysis
Among the CK1, T1, and T2 seed samples, 14 genes related to the PEG-6000 priming regulation of aging seed vigor were selected for qRT-PCR validation, using Actin as the internal reference gene.Primer Premier 5.0 was utilized for primer design, while the Vazyme RT-PCR kit (Q341) was employed.The qRT-PCR reaction mixture (25 uL) com-prised sybrGreen qPCR master mix (12.5 µL), reaction primers (10 µ mol/L) 0.5 µL each, cDNA templates (2 µL), and ddH 2 O (9.5 µL).The qRT-PCR reaction program involved an initial step at 95 • C for 2 min, followed by 40 cycles of 95 • C for 10 s and 60 • C for 40 s.Gene expression was assessed using the 2 −∆∆Ct method, Pearson's correlation coefficient method was employed for calculating the correlation coefficient, and data processing was performed as described previously [35].

Determination of SOD, POD, and GST Activity
The SOD, POD, and GST activities were assessed in fresh seed samples after 24 h of water absorption.SOD activity was determined through the nitrogen blue tetrazole photoreduction method [36], POD activity using the guaiacol method [37], and GST activity using a kit from Boxbio.

Auxin, Brassinolide, and Lignin Content Determination
After 24 h of water absorption, 0.2 g of fresh seed samples were ground in liquid nitrogen.High-performance liquid chromatography (HPLC) was used to measure IAA and BR levels [38].Ten days post sprouting, the epicotyl of the seedlings was dried at 65 • C and immediately ground.The lignin content was quantified using a lignin extraction kit from Boxbio, following the kit instructions.

Statistical Analysis of Data
One-way analysis of variance with the least significant difference (LSD) as a test at a 95% confidence interval was applied to assess the significant differences between treatments by using SPSS 22.0 (IBM, Chicago, IL, USA), and analysis results were illustrated using the Graph Pad-Prism and TB tools.

Effects of Different Concentrations of PEG-6000 Priming on Seed Vigor and Seedling Biomass of Aged Soybean
To explore the optimal concentration of PEG-6000 priming for enhancing the vigor of aged seeds, the soybean seeds were primed using varying PEG-6000 concentrations for 24 h.The results indicate that aging treatment weakened seedling growth (Figure 1A).However, priming with PEG-6000 significantly improved the growth of aging seeds compared to non-priming aging seeds.Hydro priming had no significant impact on seedling growth.Among the PEG-6000 priming treatments, the 30% concentration yielded the best results, followed by 20%, with no discernible difference between the 40% concentration treatment and the 30% PEG treatment.Statistical analysis involving germination rate, root length, seedling length, fresh weight, and dry weight demonstrated that the 30% PEG-6000 concentration priming treatment surpassed the 20% PEG-6000 priming, hydro priming, and non-priming aging treatment in all parameters, except it did not significantly differ from the 40% PEG-6000 concentration treatment (Figure 1B-G).Furthermore, the 20% PEG-6000 priming exceeded hydro priming and non-priming aging seeds, with no significant difference observed between hydro priming and non-priming aging treatment.Under the 30% PEG priming treatment, aged seeds exhibited a root length of 13.7 cm, seedling length of 11.8 cm, fresh weight of 0.94 g, dry weight of 0.19 g, and germination rate of 87.3%.The statistical analysis of the vigor index (Figure 1G) showed the highest vigor index (22.6)at 30% PEG-6000 concentration, the highest of all.These findings underscore decreased seed vigor post-aging treatment, while PEG-6000 priming effectively enhances the vigor of aged soybean seeds.Hydro priming, on the other hand, has limited efficacy in improving the vigor of aged soybean seeds.

Effects of PEG-6000 Priming at Different Duration on Seed Vigor and Seedling Biomass of Aged Soybean
To investigate the optimal duration of PEG-6000 priming for enhancing the vigor of aged seeds, aging soybean seeds underwent treatment with 30% PEG-6000 for varying durations, and the effects on seed vigor and seedling biomass were studied, and its results are shown in Figure 2A.The growth of seedlings primed by 30% PEG-6000 for 24 h was significantly better than those primed with 30% PEG-6000 for 12 h, but there was no significant difference from those primed with 30% PEG-6000 for 36 h.Statistical analysis encompassing germination rate, root and seedling length, and fresh and dry weight revealed that the 30% PEG-6000 priming for 24 h yielded significantly superior outcomes compared to 12 h priming, no priming, and was not significantly different from the 40% PEG-6000 priming treatment (Figure 2B-G).Additionally, the vigor index peaked at the 24 h mark for 30% PEG-6000 priming (Figure 2G).Collectively, these findings affirm that 24 h priming with 30% PEG-6000 significantly improved the vigor compared to non-priming aging seeds, thereby representing the optimal treatment concentration and duration.

Transcriptome Sequencing Analysis and Screening of Differentially Expressed Genes (DEGs)
To comprehensively investigate the regulatory mechanism of PEG-6000 priming on germination activity in aged seeds, transcriptome sequencing was conducted across four seed groups: newly harvested seeds (CK0), aged seeds (CK1), and PEG-6000 priming treatments for 12 h (T1) and 24 h (T2), resulting in a yield of 68.8 Gb of clean transcriptome sequencing data with a Q30 base percentage > 93.5%.Alignment with the soybean reference genome revealed a read comparison efficiency of over 96.3% for each sample.Principal component analysis and correlation analysis were employed to assess the four sample groups, confirming significant variance between them.The variance of PC1 and PC2 are 59% and 23.1%, respectively (Figure 3A), and the range of correlation coefficients was between −1 and 1, and the correlation coefficients between samples were >0.875 (Figure 3B).These indicate good repeatability in the materials and the reliability of the data in each group.After adjusting p < 0.05 and |log 2 Foldchange| ≥ 1 as the threshold, the difference between the samples was considered significant.In the CK0-CK1 comparison, 4430 DEGs were identified (Figure 3C), comprising 2007 up-regulated and 2423 down-regulated genes.Similarly, in the CK1-T1 comparison, 3176 DEGs were observed, with 1455 up-regulated and 1721 down-regulated genes.For the CK1-T2 comparison, 4588 DEGs emerged, featuring 1697 up-regulated and 2891 down-regulated genes.Notably, the prolongation of PEG-6000 priming duration correlated with an increase in up-regulated and down-regulated DEGs, highlighting the potential of PEG-6000 to induce differential gene expression associated with seed germination vigor.Further refinement focused on CK1-vs-T1 and CK1-vs-T2 comparisons.Venn diagrams (Figure 3D) were utilized to identify specific DEGs influenced by PEG-6000 priming.Notably, 1541 DEGs were exclusively expressed in CK1-vs-T1, while 2953 DEGs were exclusive to CK1-vs-T2, yielding a combined total of 1635 DEGs.These specific DEGs were attributed to PEG-6000-primed genes rather than variations resulting from different priming durations.

qRT-PCR Analysis of Some DEG Expression Patterns
To verify the accuracy of RNA-seq results, the expression patterns of 14 randomly selected DEGs were validated in CK1, T1, and T2 treatments (Figure 4).The gene change trend was consistent with the RNA-seq results, with the correlation coefficient between the results (0.815) showing a strong correlation.

GO and KEGG Analysis of These DEGs Primed by PEG-6000
To dissect the function of the DEGs, Metabolic pathway enrichment analysis was conducted on the common DEGs using GO and KEGG.GO analysis encompassed three categories: biological metabolism, cellular components, and molecular functions (Figure 5, Table S1).Regarding cellular components, DEGs were primarily concentrated in cells, cell parts, organelles, and membranes, with enrichment in metabolic processes, monomer processes, cellular processes, and responses to stimuli within the biological metabolism.In the context of molecular functions, enrichment was primarily observed in catalytic activity, followed by binding, antioxidant activity, and nucleic acid protein transcription factor activity.Notably, certain genes were not confined to a single category.
KEGG enrichment analysis revealed the significant enrichment of DEGs in metabolic pathways, secondary metabolic pathways, carbon metabolism, and phenylpropanoid metabolism post PEG-6000 priming (Figure 6, Table S2).These pathways are potentially associated with the PEG-6000 priming treatment.Notably, amino acid metabolism, antioxidant enzyme activity, and plant hormone metabolism are also implicated, suggesting their involvement in enhancing aging soybean seed vigor through PEG-6000 priming, thereby improving aging soybean seed vigor.

PEG-6000 Priming Induced Gene Expression in Carbon Metabolism, Glyoxylic Acid, and Dicarboxylic Acid Metabolism Pathways
To elucidate the effect of PEG-6000 priming on the gene regulatory network governing soybean seed germination vigor during aging, we identified common DEGs associated with carbon metabolism, glyoxylate metabolism, and dicarboxylic acid metabolism (Figure 7, Table S3).After aging treatment, gene expression analysis showed that except Glyma.12G015100 and Glyma.12G015300(encoding the 8HGO protein) and Glyma.19G111000(encoding GDH1 protein) and Glyma.07G059800(encoding GLYR1 protein), which were down-regulated, nearly all differential genes linked to carbon metabolism, glyoxylic acid, and dicarboxylic acid metabolism exhibited up-regulation.Notably, Glyma.14G042000(encoding At5g42250 protein) displayed a 4.2-fold up-regulation, suggesting heightened energy demand for seed germination post-aging treatment.Upon priming the aging seed with 30% PEG-6000, gene expression changes were observed.Glyma.12G015100 and Glyma.12G015300encoding the 8HGO protein, along with MSTRG.9897(encoding GAPC) and Glyma.19G111000(encoding GDH1), were up-regulated.Conversely, other genes implicated in carbon metabolism, glyoxylate, and dicarboxylic acid pathways experienced down-regulation.Notably, gene Glyma.10G293500encoding TKL-1 protein displayed a 2.5-fold reduction under T1 treatment and a 3.5-fold reduction under T2 treatment post PEG-6000 priming, indicating lowered energy demand for promoting aging seed germination.In essence, PEG-6000 priming facilitated seed germination during aging.

PEG-6000 Priming Enhanced Gene Expression in the Lignin Biosynthesis Pathway
To construct the metabolic pathways of phenylalanine leading to lignin and flavonoid formation, common DEGs were analyzed (Figure 8).After seed aging treatment, the expression of five peroxidase-encoding genes (Glyma.14G201700,Glyma.11G049600,Glyma.13G306900,Glyma.03G007700, and Glyma.04G059600) was down-regulated.This contrasts significantly with their up-regulated state after 30% PEG-6000 priming treatment.Particularly, Glyma.03G007700exhibited a 5.6-fold and 7.2-fold increase under T1 and T2 treatments (Table S4), respectively.Peroxidase drives the conversion of coumarin alcohol to hydroxyphenyl lignin, facilitating embryo emergence from aged seed coats.This likely underlies the enhanced germination activity of aged soybean seeds due to PEG-6000 priming.In the phenylalanine-to-flavonoid metabolic pathway, genes associated with flavonoid synthesis showed reduced expression.PEG-6000 priming treatment is believed to augment phenylalanine metabolism, favoring lignin over flavonoid production.

PEG-6000 Priming Enhanced the Gene Expression of Antioxidant Enzymes
To reveal the effect of PEG-6000 priming on the gene regulatory network governing ROS. common DEGs were analyzed (Figure 9).ROS remain inactive in dry seeds but become active on the mitochondrial plasma membrane post-seed imbibition.ROS functions as a signaling molecule, regulating seed germination.Although excessive ROS accumulation has a toxic effect on seeds and inhibits seed germination, it can be eliminated by plant antioxidant systems [39].PEG-6000 priming up-regulated genes (Glyma.10G193500,Glyma.02G024600,Glyma.03G176300, and MSTRG.13953)encoding SOD and GST enzymes.Following aging treatment, Glyma.10G193500,Glyma.03G176300, and MSTRG.13953exhibited insignificant up-regulation, which significantly changed after PEG-6000 priming.GST-encoding genes Glyma.03G176300 and MSTRG.13953demonstrated significant upregulation post PEG-6000 priming.The SOD-encoding gene Glyma.10G193500showed 2.6-fold up-regulation under T1 and 5-fold under T2 treatment (Table S5).These findings indicate that PEG-6000 priming enhances antioxidant enzyme gene expression, thereby improving germination vigor in aged soybean seeds.

PEG-6000 Priming Treatment Increased the Enzyme Activity, Hormone Content, and Lignin Content of Aged Soybean Seeds
To investigate the effect of PEG-6000 on gene expression regulation during germination of aged soybean seeds, the enzyme, hormone, and lignin levels were measured in CK1, T1, and T2 samples (Figure 10A-F).Priming with PEG-6000 enhanced the SOD, POD, and GST enzyme activities, along with IAA, BR, and lignin contents, as compared to non-primed aged soybean seeds.These findings align with the observed gene expression trends (Figures 1, 2, 8 and 9).Notably, the T2 treatment exhibited significantly higher SOD and POD activities, as well as BR and lignin contents, than the T1 treatment.Specifically, under T2 treatment, SOD activity reached 125.8 U/g, POD activity was 72.0 U/g, GST activity measured 61.2 nmol/min/mL, IAA content was 74.8 ng/g, BR content was 6.2 ng/g, and lignin content was 88.4 mg/g, which was significantly higher than CK1.These results underscore the efficacy of PEG-6000 priming in enhancing aged soybean seed vitality through increased antioxidant enzyme activity, the modulation of plant hormone metabolism, and heightened lignin content.

Discussion
Seed quality significantly influences seedling growth and crop yield.However, global industrialization, environmental pollution, and reduced arable land have severely impacted crop seedling environments.Enhancing seed quality not only boosts seed resilience against harsh stressors but also facilitates rapid, uniform, and robust emergence.This, in turn, regulates crop growth and developmental timing and ultimately elevates crop yield [46,47]. Seed priming, or osmotic regulation, optimizes germination for diverse seed batches, enabling "inferior" seeds to catch up with their "superior" counterparts before germination.Previous studies indicate that seed priming

Discussion
Seed quality significantly influences seedling growth and crop yield.However, global industrialization, environmental pollution, and reduced arable land have severely impacted crop seedling environments.Enhancing seed quality not only boosts seed resilience against harsh stressors but also facilitates rapid, uniform, and robust emergence.This, in turn, regulates crop growth and developmental timing and ultimately elevates crop yield [46,47]. Seed priming, or osmotic regulation, optimizes germination for diverse seed batches, enabling "inferior" seeds to catch up with their "superior" counterparts before germination.Previous studies indicate that seed priming enhances germination vigor [48][49][50][51].This study's findings demonstrate a significant enhancement in germination vigor and growth biomass of aged soybean seeds through seed priming.
PEG, a high molecular polymer, finds widespread use in production.PEG osmotic regulation emerges as a novel technique for enhancing seed vigor.Nevertheless, priming durations and concentrations vary across different crops [33,52,53].In cultivation, PEG holds promise as a novel agent to enhance aged seed vigor, corroborated by numerous studies [54,55].This investigation contrasts the effects of priming concentrations (20%, 30%, and 40% PEG-6000) during a 24 h treatment.The optimal treatment concentration was identified as 30% PEG-6000.Subsequent research using this concentration focused on determining the optimal initiation time.Comparative analyses across treatment durations (12 h, 24 h, and 36 h) affirmed that 30% PEG-6000 priming for 24 h represented the optimal condition, consistent with prior studies [55].This aligns with the seed germination water absorption principle, suggesting that seeds with high vigor complete functional repairs before germination, positioning them for optimal emergence.
As a signaling molecule, ROS is implicated in seed germination.It may hinder ABA transport from cotyledons to embryos, thus promoting germination [62].Alternatively, it can stimulate GA hormone signals to facilitate seed germination [63].However, excessive ROS accumulation can be toxic to seeds and impede germination.In aging seeds, reduced molecular oxygen leads to increased ROS due to a disrupted balance between ROS generation and elimination.To counter ROS's adverse effects on germination, seeds have evolved a complex antioxidant defense system encompassing antioxidant metabolites (ascorbic acid, glutathione, and flavonoids) and enzymes (SOD, POD, glutathione sulfhydryl transferase, glutathione peroxidase, ascorbic acid peroxidase) [64,65].SOD scavenges auto-aerobic an-ions and catalyzes O 2 and H 2 O 2 formation [66].Glutathione sulfhydryl transferase reduces glutathione to form conjugates, degrading harmful substances [67].This study found the significant up-regulation of genes: Glyma.10G193500(encoding SOD), Glyma.02G024600,Glyma.03G176300, and MSTRG.13953(encoding GST) after PEG-6000 priming.Notably, GST protein-encoding genes (Glyma.03G176300and MSTRG.13953)displayed significant up-regulation with PEG-6000 priming.The gene Glyma.10G193500(encoding SOD) exhibited 2.6-fold up-regulation under T1 treatment and 5-fold under T2 treatment.Furthermore, superoxide dismutase and glutathione sulfhydryl transferase activities were assessed, revealing significantly higher enzyme activity with priming treatment compared to non-priming treatment.This indicates that priming treatment enhances aged soybean seed activity by boosting antioxidant enzyme activity.In previous research, PEG priming has been verified to enhance seed tolerance to abiotic stress through H 2 O 2 signal transduction [32], while in this study, it may also be involved in this signal transduction.Further research is essential to confirm this hypothesis.
Peroxidase, a cysteine-dependent enzyme, catalyzes hydrogen peroxide into water [68], which is pivotal in hydroxyphenyl lignin pathway formation under environmental stress in prior studies [69].In this work, five peroxidase genes (Glyma.14G201700,Glyma.11G049600,Glyma.13G306900,Glyma.03G007700, and Glyma.04G059600) were upregulated in the phenylalanine-lignin biosynthesis pathway.They convert coumaric alcohol to lignin, promoting embryo breakthrough (germination) of the seed coat.This mechanism may underlie the enhanced germination via the PEG-6000 priming of aged soybean seeds.Further peroxidase activity and lignin content measurements indicated significantly higher values for PEG-primed compared to un-primed aged seeds.Peroxidase likely serves as the key enzyme for PEG priming's invigoration of aging soybean seeds.
Ascorbate peroxidase and flavonoids also impact seed antioxidant processes and aging regulation.Within the phenylalanine-flavonoid pathway, genes Glyma.03G181600 and Glyma.03G181700(encoding PAL protein) and Glyma.17G064400(encoding 4CL protein) were down-regulated, suggesting higher lignin than flavonoid production.Flavonoid metabolism pathway genes are predominantly down-regulated, indicating PEG priming suppresses flavonoid-related genes (Figure 11, Table S7).Interestingly, ascorbic acid peroxidase genes were also down-regulated.The improvement in aging soybean seed vigor by PEG priming may not rely on the ascorbic acid and flavonoid pathway, or this pathway's role might be specific to the priming-to-drying stage rather than seed coat breakthrough.Further research is essential to confirm this hypothesis.
Based on the above experimental findings, PEG priming treatment appears to retain the original "memory" of aged seeds, requiring minimal energy metabolism for germination.Simultaneously, it triggers ROS signaling molecules, promoting IAA and BR biosynthesis, while suppressing abscisic acid biosynthesis-related gene expression.ROS scavenging activates within the seeds, enhancing SOD, POD, and glutathione sulfhydryl transferase activities, and promoting the conversion of coumaryl alcohol into hydroxy-phenyl lignin, facilitating aged seed germination.
Prior research demonstrated that PEG priming enhances aged soybean seed vigor through the modulation of carbon metabolism, reactive oxygen species scavenging, hormone signaling, and lignin synthesis.This underscores the profound importance of PEG priming technology for the growth and morphological establishment of aging soybean seedlings, with practical implications for production.

Conclusions
The polymer PEG-6000 is able to enhance aging seed vigor, which has been confirmed by many scholars [21][22][23][24][25].However, the effect of PEG-6000 on aging soybean seed vigor and its molecular regulatory mechanism are still unclear.To explore the impact of PEG-6000 priming on aged soybean seeds' vigor, the morphological, physiological, biochemical, and transcriptomic analysis were performed.The main findings are as follows: (i) 30% PEG-6000 priming for 24 h can significantly improve the germination vigor of aged soybean seeds; (ii) the PEG priming treatment of aged seeds suppressed carbon metabolism, which facilitated energetically efficient germination; (iii) the PEG priming treatment of aged seeds stimulated lignin biosynthesis with the up-regulation genes of peroxidase; (iv) the PEG priming treatment of aged seeds boosted IAA and BR synthesis and reduced the expression of abscisic acid biosynthesis-related genes; and (v) the PEG priming treatment of aged seeds enhanced the ROS system with the up-regulation genes of SOD, POD, and GST.These results suggest that PEG-6000 priming enhanced aged soybean seed vigor through the modulation of carbon metabolism, reactive oxygen species scavenging, hormone signaling, and lignin synthesis.It is worth noting that five peroxidase-encoding genes displayed significant up-regulation, promoting the conversion of coumaryl alcohol to hydroxy-phenyl lignin, a probable catalyst for augmented seed vigor.Further research is needed to investigate the functions of these genes.

Figure 1 .
Figure 1.Phenotype differences among new seeds (SG), aged seeds (AA), aged seeds in hydro priming (HP), and different concentrations (20%, 30%, 40%) of the PEG-6000 priming (PEG20P, PEG30P, PEG40P) for 24 h.(A) refers to the growth state of germinated seedlings under different treatments.(B-G) refer to root length, seedling length, germination rate, seedling dry weight, seedling fresh weight, and germination vigor index under different treatments, respectively.The errors in the figure represent the standard deviation (SD).Lowercase letters indicate a significant difference of p < 0.05.

Figure 2 .
Figure 2. Phenotypic differences among aged seeds (AA) and aged seeds primed with 30% PEG-6000 for 12 h (PEG30P-12 h), 24 h (PEG30P-24 h) and 36 h (PEG30P-36 h).(A) refers to the growth state of germinated seedlings under different treatments.(B-G) refer to root length, seedling length, germination rate, seedling dry weight, seedling fresh weight, and germination vigor index under different treatments.The error in the figure represents the standard deviation (SD).Lowercase letters indicate a significant difference of p < 0.05.

Figure 3 .
Figure 3. Principal component analysis (PCA), correlation coefficient, and number of DEGs among the different treatments.(A,B) represent the PCA and Pearson's correlation coefficients of three biological replicates of four treatments, respectively.(C) represents the DEG statistics of the different comparison combinations; (D) represents the DEG Wayne diagram of the different comparison combinations.

Figure 4 .
Figure 4. Expression pattern verification of some DEGs of CK1, T1, and T2 and correlation analysis of RNA-seq.The gene expression level is represented by the FPKM value.

Figure 5 .
Figure 5. GO enrichment analysis of common DEG.

Figure 6 .
Figure 6.Scatterplot of the top 30 metabolic pathways in the KEGG enrichment analysis of common DEGs.

Figure 7 .
Figure 7. DEGs of carbon, glyoxylic acid, and dicarboxylic acid metabolism of the standard germination test (CK0), aging treatment (CK1), 30% PEG-6000-induced aging seeds for 12 h (T1), and 30% PEG-6000-induced aging seeds for 24 h (T2).The circle inside represents the corresponding pathway of the gene, with different colors representing different genes and different positions indicating that the gene belongs to carbon, glyoxylic acid, and dicarboxylic acid metabolism.The outermost layer represents the gene name.Different layers represent different gene expression levels.The gene expression in the figure is the FPKM value of the exon model.

Figure 9 .
Figure 9. DEGs involved in plant hormones and antioxidant pathways of standard germination test (CK0), aging treatment (CK1), 30% PEG-6000-induced aging seeds for 12 h (T1), and 30% PEG-6000-induced aging seeds for 24 h (T2).The left side represents different genes, different positions represent different pathways, with the color mark located on the right side, and the gene expression level is represented by the FPKM value.

Agronomy 2023 , 21 Figure 10 .
Figure 10.Plant hormone, lignin, and antioxidant enzyme contents of aging treatment (CK1); 30% PEG-6000 induced aging seeds for 12 h (T1); and 30% PEG-6000 induced aging seeds for 24 h (T2).(A-F) refer to the content of SOD enzyme, POD enzyme, GST enzyme, IAA, BR, and lignin contents under different treatments, respectively.The error in the figure represents the standard deviation (SD).Lowercase letters indicate a significant difference of p< 0.05.

Figure 10 .
Figure 10.Plant hormone, lignin, and antioxidant enzyme contents of aging treatment (CK1); 30% PEG-6000 induced aging seeds for 12 h (T1); and 30% PEG-6000 induced aging seeds for 24 h (T2).(A-F) refer to the content of SOD enzyme, POD enzyme, GST enzyme, IAA, BR, and lignin contents under different treatments, respectively.The error in the figure represents the standard deviation (SD).Lowercase letters indicate a significant difference of p< 0.05.

Figure 11 .
Figure 11.DEGs were involved in flavonoid biosynthesis and ascorbic acid metabolism pathways of the standard germination test (CK0), aging treatment (CK1), aging seeds induced by 30% PEG-6000 for 12 h (T1), and aging seeds induced by 30% PEG-6000 for 24 h (T2).The left side of the figure represents the different genes (different positions representing different pathways), with the color code located on the right side and the gene expression level represented by the FPKM value.