Transcriptomic insights into soybean seed deterioration in uncontrolled storage

ABSTRACT

Soybean, the main agricultural commodity of Brazil, is sensitive to deterioration during storage due to its high fatty acid content and fragile seed coat, especially under tropical conditions where high temperature and humidity accelerate this process. The objective of this study is to investigate the mechanisms that are altered during the storage of soybean seeds under uncontrolled environmental conditions. For this purpose, RNA-seq libraries were generated from freshly harvested soybean seeds (FHS) and seeds stored under uncontrolled conditions (UES). The total RNA from seeds of the cultivar BRS 413 was extracted and sequenced for gene expression analysis. Relative expression indicated 325 genes repressed in UES and 150 induced when comparing the genes in stored seeds to freshly harvested ones. Genes related to protein folding, carbohydrate metabolism, lipids, phytohormones, and transcription factors showed a significant reduction in their expression during the storage. Among the genes uniquely expressed in UES, alanine-glyoxylate transaminase (GGAT), cysteine synthase (CSase), aldehyde dehydrogenase (ALDH), thioredoxin peroxidase (Peroxiredoxin), cytochrome P450 and protein phosphatase 2C (PP2C) stood out for making part of protective mechanisms against oxidative stress in plants.

Index terms:
Glycine max L.; RNA-seq; seed viability

RESUMO

A soja, principal commodity agrícola do Brasil, é sensível à deterioração durante o armazenamento devido ao seu alto teor de ácidos graxos e tegumento frágil, especialmente sob condições tropicais onde altas temperaturas e umidade aceleram esse processo. O objetivo deste estudo é investigar os mecanismos que são alterados durante o armazenamento de sementes de soja sob condições ambientais não controladas. Para isso, foram geradas bibliotecas de RNA-seq a partir de sementes de soja recém-colhidas (FHS) e sementes armazenadas em condições não controladas (UES). O RNA total das sementes da cultivar BRS 413 foi extraído e sequenciado para análise de expressão gênica. A expressão relativa indicou 325 genes reprimidos em UES e 150 induzidos ao comparar os genes nas sementes armazenadas com as recém-colhidas. Genes relacionados ao dobramento de proteínas, metabolismo de carboidratos, lipídios, fitohormônios e fatores de transcrição mostraram uma redução significativa em sua expressão durante o armazenamento. Entre os genes exclusivamente expressos em UES, alanina-glioxilato transaminase (GGAT), cisteína sintase (CSase), aldeído desidrogenase (ALDH), peroxidase tioredoxina (Peroxiredoxina), citocromo P450 e proteína fosfatase 2C (PP2C) se destacaram por fazerem parte de mecanismos de proteção contra o estresse oxidativo em plantas.

Termos de indexação:
Glycine max L.; RNA-seq; viabilidade de sementes

INTRODUCTION

Soybeans are the main agricultural commodity in Brazil, with a production of 147,684.8 million tons in a cultivated area of more than 45,733.2 thousand hectares (CONAB, 2024). The storability of soybean seeds, that is, their capacity to maintain quality and viability during storage, is a determining factor for the proper establishment of plants in the field (Schuch et al., 2009; Rossi et al., 2017; Ramtekey et al., 2022). Legume crops, such as soybeans, are susceptible to deterioration during storage due to their high fatty acid content and their anatomy characterized by the presence of a fragile seed coat (Rao et al., 2023). Lipid peroxidation and the change in fatty acid content are initiated by free radicals that form due to stress conditions in the seed, which alters the structure of unsaturated acids, causing damage to cell membranes (El-Maarouf-Bouteau, 2022; Li et al., 2022).

The seed deterioration process is inevitable and irreversible, influenced by intrinsic and extrinsic factors and perceived by cytological, physiological, biochemical and physical changes that are associated with loss of quality and viability (Ebone et al., 2019). Deterioration begins when the seed reaches physiological maturity (before harvest) and continues during processing and storage at a rate influenced by genetics, production, and environmental factors (Abati et al., 2022, Rao et al., 2023). Deterioration results in reductions in germination rate and emergence in the field, increase in abnormal seedlings, loss of vigor and, ultimately, seed death. Internally, the process is marked by loss of membrane integrity, changes in the structure of macro molecules, enzymatic degradation, DNA damage and RNA degradation, impairing protein synthesis (El-Maarouf-Bouteau, 2022).

In tropical countries, the dynamics of seed deterioration are more relevant, as storage under uncontrolled environmental conditions, considering high temperature and humidity, compromises seed quality, reducing the final plant stand in the field (Monira et al., 2012; Zuchi et al., 2013; Abati et al., 2022). Commercially, seeds are stored in raffia bags, called “big bags”, and although during storage seed companies and producers already make use of refrigeration under favorable conditions, the seed is still exposed to conditions during transport to the property and during the sowing period (Coradi et al., 2020).

In this context, transcriptome analysis of soybean seeds during storage under uncontrolled conditions can be a powerful tool for advancing knowledge about the biological processes associated with the post-harvest deterioration process during the off-season storage period (Jones and Vodkin, 2013; Pereira-Lima et al., 2017; Molinari et al., 2021a; Kafer et al., 2023; Molinari et al., 2023). Understanding the genes activated during storage under non-ideal conditions can help in the design of genetic strategies aimed at obtaining more tolerant genotypes.

This study proposes a detailed investigation on the molecular bases that are altered during the storage of soybean seeds under uncontrolled environment conditions.

MATERIAL AND METHODS

To obtain the biological material for RNA-Seq analysis, seeds were multiplied in a greenhouse, using the cultivar BRS 413 RR, of yellow seed coat, considered moderately resistant to storage (Abati et al., 2021; Abati et al., 2022). The seeds were wrapped in germination paper previously moistened with 30 g of water. Then, they were incubated in a germination chamber with an alternating photoperiod of 12 hours of light and 12 hours of dark, under a temperature of 28 ± 1 °C and relative humidity of 100%, for a period of 4 days. After germination, two seedlings with uniform primary root were transferred to each pot (8 L), which contained a mixture of 1:1 substrate (fertilized soil and washed sand), in a total of 100 pots. The experiment was carried out in a greenhouse under conditions of short days (10 h of light/14 h of dark) at 28 ± 2 °C and optimal irrigation conditions.

The seeds were harvested when the plants reached full maturity, threshed manually, and then homogenized to form a 500 g working sample. The samples were divided into two treatments: freshly harvested seeds (FHS) that were stored in ultra-freezer (-80 °C) and seeds stored for 6 months under uncontrolled conditions (UES), with 3 replicates of each treatment. After the storage period, both seeds were kept at -80 °C until the moment of extraction.

Samples of whole mature seeds were used for total RNA extraction. The samples were macerated in liquid nitrogen and the total RNA was extracted using the Concert Plant RNA reagent kit (Invitrogen, California, USA) according to the manufacturer’s specifications. RNA quantification was performed in a NanoDrop spectrophotometer, according to the following quality and purity parameters: concentration > 600 ng.μL^-1, 260/280 ratio ranging from 1.8 to 2.0, and 260/230 ratio ≥ 2.0.

The RNA was treated with the turbo DNAse-free kit for removal of remaining genomic DNA (Invitrogen, California, USA). RNA integrity was assessed by electrophoresis on 1% (w/v) agarose gel stained with ethidium bromide (1 μg.mL^-1) (Sambrook et al., 1989). The high-quality total RNA samples were sent for sequencing at the University of Georgia (Georgia Genomics Facility - GGF, USA). Before the samples were sequenced, they were evaluated in the Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.), and only samples with RNA integrity number (RIN) ≥ 7.00 were used to synthesize the mRNA-Seq libraries. The libraries were synthesized using the Illumina TruSeq™ SBS v5 Poly-A kit on Illumina NextSeq 500 1.9 sequencing platform of 75 bp paired-end reads (Illumina, San Diego, CA, USA), with at least 1X genome coverage. Each sample was sequenced in triplicate, yielding 6 mRNA libraries (3 libraries for FHS, 3 libraries for UES).

The quality of the raw fragments (FASTQ) was evaluated using the FastQC v.0.11.5 software before and after the removal of adapters and low-quality sequences, and one of the replicates of each treatment was removed from the analysis because it did not meet the quality standards. Fragment cleaning was performed using the Trimmomatic software, version 0.36, standardizing sections every four nucleotides at the ends of the sequences that had a quality score lower than 30 (Phred Quality Score, Q ≥ 30).

The fragments were aligned by the HISAT2 v.2.1.0 software using the soybean genome Wm82.a2.v1 as reference. Then, PCR artifacts from the Illumina sequencing were removed using the Samtools v.1.5 software. Transcripts were assembled using the Stringtie v.1.3.3 software and the relative expressions were obtained from the EdgeR v.3.22.3 software via RStudio v.3.5.1. The differentially expressed genes were obtained by comparing FHS and UES. Genes with Log2 Fold Change (Log2FC) values ≤ − 1 and ≥ + 1 with a false positive rate (FDR) ≤ 0.05 and positive logTPM (Transcripts per million) were considered differentials. Annotation of the genes was performed using the Phytomine tool. The bioinformatics analyses were carried out according to Kafer et al. (2023) and Molinari et al. (2021b). Analysis of gene enrichment and gene ontology was performed by the ShinyGO software based on information from the KEGG pathways database.

RESULTS AND DISCUSSION

Regarding the differential expression analysis, when comparing the libraries generated in the UES treatment with the libraries corresponding to the FHS treatment, 475 differentially expressed genes were identified, 150 of which were upregulated and 325 downregulated. The genes were considered modulated when they contained at least 3 TPMs in one of the repeats, and absent when they had TPM < 3. Also, genes without annotation were eliminated from the analysis. Thus, 24 genes were considered only expressed in the UES treatment and 12 genes only expressed in the FHS treatment (Supplementary file).

Among the genes solely expressed in UES, the genes alanine-glyoxylate transaminase (GGAT), cysteine synthase (CSase), aldehyde dehydrogenase (ALDH), Thioredoxin peroxidase (Peroxiredoxin), Cytochrome P450 and Protein phosphatase 2C (PP2C) stand out for being part of protective mechanisms against oxidative stress in plants (Kendziorek et al., 2012; Bela et al., 2015; Tiwari et al., 2017; Du et al., 2022; Yang et al., 2023). Oxidative stress during seed storage occurs due to accumulation of reactive oxygen species that cause damage to nucleic acids, proteins, and polyunsaturated fatty acids (Rao et al., 2023). In addition, reduced protection mechanisms against reactive oxygen species (ROS) has been related to loss of seed viability (Lin et al., 2022). The activity of these genes may be associated with the stress response present during storage as a protective mechanism.

The Kegg pathways of modulated genes (TPM≥3) were manually identified, and protein processing pathways and chaperones, hormone signaling genes, transcription factors, carbohydrate and energy metabolism, and lipid metabolism were associated (Supplementary file). These pathways account for about 37% of downregulated genes and about 15% of upregulated genes in UES treatment compared to FHS treatment (Figure 1). In both treatments, the genes that are not present in this classification represent metabolic pathways and processes that did not show sufficient numbers to be included in the analysis, representing 63% and 85% of the genes in FHS and UES, respectively.

Figure 1
Expression pattern of A) downregulated and B) upregulated genes in the UES treatment compared to the FHS treatment, for pathways related to deterioration and quiescence processes in soybean seeds C) Number of downregulated and upregulated genes in soybean seed during storage.

In general, a reduction in the gene expression profile was observed in soybean seeds after storage in all observed pathways (Figures 2A and B; 3A and B; 4A and B; 5A and B; 6A and B). This is attributed to reduced metabolism in the seed, which tends to go into quiescence soon after maturation and harvesting. In addition, the decrease in the expression of seed protection systems may be associated with deterioration due to storage in an uncontrolled environment.

Figure 2
Comparative analysis of gene expression of chaperones and proteins associated with protein folding in stored (UES) versus freshly harvested (FHS) soybean seeds, (A) Heatmap containing the transcript counts (Transcripts per million) calculated from the average of two replicates. (B) Logarithmic changes (Log2FC) with statistical significance (FDR=0.05).

Figure 3
Comparative analysis of gene expression of hormone biosynthesis and signaling in stored (UES) versus freshly harvested (FHS) soybean seeds, (A) Heatmap containing the transcript counts. (B) Logarithmic changes (Log2FC) with statistical significance (FDR=0.05).

Figure 4
Comparative analysis of gene expression of transcription factors in stored (UES) versus freshly harvested (FHS) soybean seeds, (A) Heatmap containing the transcript counts (Transcripts per million) calculated from the average of two replicates. (B) Logarithmic changes (Log2FC) with statistical significance (FDR=0.05).

Figure 5
Comparative analysis of gene expression of carbohydrate biosynthesis in stored (UES) versus freshly harvested (FHS) soybean seeds, (A) Heatmap containing the transcript counts (Transcripts per million) calculated from the average of two replicates. (B) Logarithmic changes (Log2FC) with statistical significance (FDR=0.05).

Figure 6
Comparative analysis of gene expression of lipid biosynthesis in stored (UES) versus freshly harvested (FHS) soybean seeds, (A) Heatmap containing the transcript counts (Transcripts per million) calculated from the average of two replicates. (B) Logarithmic changes (Log2FC) with statistical significance (FDR=0.05).

Storage in tropical regions for a prolonged period of time induces stress in the seed, which can accelerate its deterioration and reduce its germination capacity (Zuchi et al., 2013; Jiamtae et al., 2022; Abati et al., 2022). However, it should be noted that, even with a reduction in expression compared to the FHS treatment, many of these genes remained highly expressed in the UES condition, as observed in Figures 2A, 3A, 4A, 5A and 6A, where the TPM values for some genes remain high, even after storage.

In the chaperone pathway and protein processing, 22 downregulated genes and 3 upregulated genes were identified (Figures 2A and 2B). HSPs are chaperones that repair proteins during deterioration under high temperatures (Arya et al., 2024). In soybeans, they are responsive to heat during flowering (Ding et al., 2020). Pereira-Lima et al. (2017), through RNA-Seq and RTqPCR, found an increase in hsps expression in seeds at full maturity compared to seeds at early maturity. Regarding proteases, they are involved in maintaining protein quality and maintaining cellular homeostasis (Adam et al., 2019). Li et al. (2024) found that Filamentation temperature-sensitive H proteins (FtsH) have a protective function against oxidative stress in Medicago truncatula.

Changes in the gene expression of phytohormone signaling pathways were observed (Figures 3A and 3B). Eleven downregulated genes and 4 upregulated genes were identified. Among the downregulated genes, there was a reduction in the signaling pathways of jasmonate, gibberellins, ethylene and auxins, being 2x less expressed than those observed in stored seeds. The GA2ox gene encodes for the enzyme gibberellin 2-oxidase responsible for the degradation of gibberellins, which are hormones that promote germination (Li et al., 2019, Xing et al., 2023). Regarding ethylene, its function was associated with breaking dormancy and promoting germination processes, acting in the regulation between GA and ABA (Ahammed et al., 2020, Zabala et al., 2020). In Petunia, loss of function of ACO (aminocyclopropane-1-carboxylic acid oxidase) results in loss of seed weight and inhibits germination (Naing et al., 2021). Regarding auxin signaling genes (IAA and TIR1), the TIR1 (Transport Inhibitor Response 1) protein acts as an auxin receptor, functioning as a facilitator in the degradation of Aux/IAA (Indole-3-Acetic Acid 1), which are repressors of auxin-responsive genes (Fendrych et al., 2018). Notably, brassinosteroid pathways were induced in UES through the expression of the brassinosteroid (BRs) signaling genes (BZR1_1 and BIN2). BIN2 and BZR1/BES1 are part of a negative signaling pathway that regulates the response of plants to BRs. This results in the suppression of BRs signaling, restricting the growth and development promoted by these hormones (Ali et al., 2022).

We identified 16 downregulated transcription factors and 7 upregulated transcription factors in relation to storage (Figures 4A and 4B). The transcription factors AP2 (APETALA2) and NAC (NAM, ATAF1/2, CUC2) were found in greater numbers with 6 and 3 copies, respectively, with a reduction in expression of up to 4.48x. Regarding the transcription factors observed, the AP2 family stood out, a family of proteins that play crucial roles in the regulation of plant development and in the response to environmental stresses. They are characterized by the presence of one or more AP2 domains, which are responsible for binding to DNA and regulating gene expression (Ali et al., 2022). As observed by Jiang et al. (2020), the overexpression of soybean AP2 genes in Arabdopsis thaliana led to early flowering and increments in size, length, and total area compared to the wild type. In rice, Youngqi et al. (2020) demonstrated that there is a greater presence of transcription factors in the initial phase of seed imbibition. Regarding the transcription factors, NAC constitute one of the largest families of transcription factors in plants, having importance in both dormancy and germination (Song et al., 2022; Shu et al., 2024). NAC have been indicated as regulators of hormonal balance between GA and ABA. In rice, Zhao et al. (2023) identified OsNAC2 as being a regulator of germination: loss of function of the gene accelerates germination, while overexpression of the gene has an inhibitory effect on seeds. In addition, the authors found that the gene acts on ABA catabolism.

The gene expression levels of genes involved in carbohydrate metabolism, cell wall formation and energy were also differentially expressed when the UES treatment was compared with FHS. In total, 22 genes related to this group were found, 17 of which were downregulated and 5 were upregulated (Figures 5A and 5B). In rice, genes related to cell wall signaling were induced during the germination process (Gigli-Bisceglia et al., 2020; Zhao et al., 2020). In soybean, these genes were induced in embryonic axes 3 to 24 after imbibition, indicating great activity of this pathway (Bellieny-Ravelo et al., 2016; Ducatti et al., 2022). Similarly, Xu et al. (2022) observed that pectinesterases act on lettuce germination. During maturation, seeds accumulate sugars and deposit them in the form of starch (Pereira-Lima et al., 2017; Khambanpati et al., 2021). The enzymes Susy (sucrose synthase), TPS (Trehalose-6-Phosphate Synthase) and glgC (Glucose-1-phosphate Adenylyltransferase) are responsible for the synthesis of sucrose, trehalose and ADP-alpha-D-glucose, respectively. In tomato, the repression of the Susy and FRK genes through RNAi resulted in smaller size and lower germination rate in the mutants compared to the wild type (Lugassi et al., 2022). Similarly, mutants for the TPS gene showed slow germination, lower cell division, and starch accumulation (Fitchner et al., 2020). Overexpression of the TPS gene caused an increase in photosynthesis and yield in Brassica napus (Yuan et al., 2024). In addition, Susy, TPS and glgC were associated with the protection against abiotic stresses (Ahmad et al., 2022, Liu et al., 2024).

Lipid metabolism and fatty acid biosynthesis were also significantly altered, with a general reduction in the number of genes present and their expression. Eleven downregulated genes and 3 upregulated genes were identified for this metabolic pathway (Figures 6A and 6B). Regarding lipid metabolism, significant changes occurred in genes, such as oleosins and lipases, suggesting that storage can affect lipid integrity and the mobilization of energy reserves. Oleosins are essential for the protection of stored fats, and a decrease in their expression may have implications for lipid quality and available energy after rehydration (Shang et al., 2020; Yuan et al., 2021). In addition, overexpression of the GmPLDγ gene increases the oil content and modulates the composition of fatty acids in Arabdopsis thaliana (Bai et al., 2020). Similarly, Liu et al. (2013) used a soybean oleosin to increase the oil content in rice. FAD2 (fatty acid desaturase 2) and ACOT (Acyl-CoA thioesterase), are responsible for the introduction of double bonds in fatty acids and hydrolysis of acyl-CoA thioesters to free fatty acids and Coenzyme A (CoA), respectively (Kalinger et al., 2020). Both showed higher expression in soybean seeds from two to six weeks after flowering (Peng et al., 2021). In addition, mutations in the FAD2 gene alter lipid profile in the plant, leading to an increase in oleic acid in the seed (Brink et al., 2014; Zhang et al., 2023). In Brassica napus, FAD2 overexpression promotes a higher germination rate and epicotyl elongation (Wang et al., 2010).

Enzymes responsible for lipid degradation were also identified, namely LOXs2 (lipoxygenase), ADH1 (alcohol dehydrogenase) and ALDH (aldehyde dehydrogenases) (Figures 6A and B). These enzymes play an important role in germination and defense against oxidative stress (Viswanath et al., 2020). In soybean and rice, loss of function of LOXs2 is associated with greater seed longevity (Huang et al., 2014). In rice, higher ADH1 activity facilitates energy production and increases germination rate (Wang et al., 2020). ALDH, on the other hand, is an enzyme associated with defense against abiotic stresses (Cao et al., 2023).

CONCLUSIONS

During the storage of soybean seeds under uncontrolled environment conditions, exclusive genes associated with protection against oxidative stresses (GGAT, CSase, ALDH, Peroxiredoxin, Cytochrome P450 and PP2C) were identified.

A reduction in the expression of genes related to chaperones, histones, hormonal pathways, lipid and carbohydrate metabolism, and transcription factors was also observed. These changes indicate a decrease in protection systems and in quiescence metabolism, highlighting the molecular impacts of storage.

ACKNOWLEDGMENTS

We thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for granting a doctoral scholarship to JMK. This research was funded by Empresa Brasileira de Pesquisa Agropecuária (Embrapa), INCT BioSyn (National Institute of Science and Technology in Synthetic Biology), CNPq (National Council for Scientific and Technological Development), and FAPDF (Research Support Foundation of the Federal District), Brazil.

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Edited by

• Editor:
  Heloisa Oliveira dos Santos

Publication Dates

• Publication in this collection
  21 Mar 2025
• Date of issue
  2025

History

• Received
  08 Aug 2024
• Accepted
  16 Dec 2024