RNA-Seq based transcriptomic analysis revealed genes associated with seed-flooding tolerance in wild soybean (Glycine soja Sieb. & Zucc.)

Highlights

• Pigmented seed-coat genotypes (SFT-tolerant) were found highly tolerant to seed-flooding stress compared to yellow seed-coat genotypes.

• Based on the results of DEGs, it has been reported that SFT-tolerant genotype is less affected by seed-flooding stress compared to the SFS-sensitive.

• Response to seed-flooding stress was mostly enriched by the terms viz., cell wall, TF antioxidant activity, catalytic and transcription factor activities as well as protein metabolism and signaling.

• Based on qRT-PCR and sequencing analysis, five genes viz., were considered as the most possible candidate genes for seed-flooding but needs further validation.

Abstract

Seed-flooding stress is one of the most important constraints that affects the yield and quality of soybean. However, very little is known about the molecular mechanisms underlying seed-flooding tolerance. Hence, the present study investigated the transcriptome profile in root tissues of two contrasting soybean genotypes viz., PI342618B/SFT-tolerant, and NN86-4/SFS-sensitive under seed-flooding stress using RNA-Seq approach. A total of 1563 and 1958 differentially expressed genes (DEGs) were identified in SFT-tolerant and SFS-sensitive, respectively suggesting that the former is less affected by flooding stress. Both Gene ontology (GO) enrichment and MapMan pathway analyses revealed that response to seed-flooding stress was mostly enriched in the DEGs that are involved in the cell wall, antioxidant activity, catalytic and transcription factor activities as well as protein metabolism and signaling. Based on gene annotation, GO enrichment and protein-protein interaction (PPI) network analysis, a total of 807 DEGs were screened from the above terms related to the stress including response to stress, defense response and response to stimulus for candidate gene prediction analysis. Out of them, 51 genes revealing significant opposite gene expression pattern between tolerant and sensitive genotypes along with GmERFVII1, GmERFVII2 & GmERFVII3 and MAPK1 were predicted as the possible candidate genes for further analysis. Using quantitative real-time RT-PCR (qRT-PCR) and sequencing analysis, five of the randomly selected ten genes viz., Glyma.01G231200, Glyma.08G083300, Glyma.06G045400, Glyma.05G215900, and Glyma.15G015100 revealed both significant opposite expression pattern and nucleotide differences, respectively between SFT-tolerant and SFS-sensitive. Hence, these genes will be of great value for breeding seed-flooding tolerance in soybean after their proper functional validation. Overall, the transcriptome profiling indicates that all the three major processes viz., biological process (BP), cellular components (CC), and molecular functions (MF) are altered in the soybean in response to seed-flooding stress. In conclusion, these findings provide in-depth insights that will be of great utility for the exploration of candidate gene functions for seed-flooding tolerance in soybean.

Introduction

Soybean (Glycine max L. Merr.) is one of the most economically important oilseed crop being rich in edible protein and oil (Hou et al., 2009). However, soybean crop is subjected to many environmental challenges such as biotic and abiotic stresses. However, in recent years, the changing global climate has led to increased occurrence of flooding events (Bailey-Serres et al., 2012). Seed-flooding or pre-germination flooding is a common abiotic stress resulting due to transient and excessive rainfall or irrigation with poor drainage during the time of seed sowing that in turn severely affects the seed germination and seedling establishment, and leading to considerable yield losses. Previous studies have reported that soybean is sensitive to flooding stress at different growth stages viz., germination, vegetative and reproductive stages (Oosterhuis et al., 1990; Sayama et al., 2009; Zhao et al., 2017). However, response to flooding stress has been extensively studied in soybean at vegetative and reproduction stages, but very limited research has been focused on understanding the mechanisms of flooding tolerance at the germination stage. Hence, efforts are required to comprehend the potential adaptive molecular mechanisms and associated genes underlying the seed-flooding tolerance in soybean.

Seed germination is an important physiological process that involves three different phases viz., rapid and passive water absorption (phase I), plateau of water absorption phase (phase II) and steady increase of water absorption (phase III) in which the radicle begins to grow (Weitbrecht et al., 2011). The phase II is also considered to be an active phase of metabolism where germination is initiated by reactivation of essential physiological and biochemical processes, such as hydrolysis, respiration, biosynthesis of macromolecules, subcellular structures, and cell elongation (Bewley et al., 2013). Moreover, germination is regulated by multiple phytohormones, such as auxin, cytokinin, ethylene, gibberellic acid, jasmonic acid, abscisic acid, and salicylic acid and by environmental conditions, including oxygen, temperature, and light (Miransari and Smith, 2014).

However, many studies have reported the adverse effects of seed-flooding stress on the seed germination and seedling establishment due to rapid water absorption, breakdown of seed structure and outflow of internal seed contents in pea (Powell and Matthews, 1978), maize (Zaidi et al., 2012), and soybean (Duke et al., 1986). In early seed-flooding stress, seed germination is adversely affected by ambient temperature, lack of oxygen, duration of flooding and seed moisture content in soybean (Tian et al., 2005; Wuebker et al., 2001). Several physiological studies reported that seed coat color, seed-coat integrity, and seed size are the essential determinants of germination in response to pre-germination flooding tolerance (Sayama et al., 2009). Pigmented seed coat has been reported to provide increased protection and tolerance to seed-flooding stress compared to a non-pigmented seed-coat. For example, black colored seed-coat has been demonstrated to exhibit enhanced tolerance to seed-flooding, whereas soybean seeds with yellow seed-coat failed to germinate after eight days of complete submergence (Hou and Thseng, 1992). Similarly, seeds with red-colored seed-coat showed greater tolerance to seed-flooding compared to white-colored seeds in wheat (Ueno and Takahashi, 1997).

Over the past few decades, molecular mechanisms related to flooding tolerance has been extensively studied in crop plants such as Arabidopsis and rice especially by rapidly discovering molecular oxygen sensing mechanism in Arabidopsis (Gibbs et al., 2011; Licausi et al., 2011). For example, group VII ethylene response factors (ERFs-VII) are considered as a key component regulating expression of flood responsive genes under low oxygen environments (Voesenek and Bailey‐Serres, 2015). Similarly, in rice two ethylene-driven transcription factors, viz., SNORKEL and Submergence‐1A promote escape and quiescence strategies, respectively, in response to flooding stress (Hattori et al., 2009; Xu et al., 2006). In Arabidopsis, group VII of ERFs (ERF-VII) viz., HRE1, HRE2, RAP2.2, RAP2.3, or RAP2.12 are found to be involved in submergence and oxygen deficiency tolerance (Licausi et al., 2011). These Arabidopsis ERF-VII are the components of the N-end rule pathway owing to the conserved N-terminal motif (MCGGAI) identified by the plant cysteine oxidase enzyme of the NERP (White et al., 2018). In addition, limited oxygen under anoxia conditions causes accumulation of reactive oxygen species (ROS) on the membrane, resulting in membrane lipid peroxidation and low antioxidant enzyme activity (Blokhina et al., 2003). Plants can exploit its antioxidant defense mechanisms by generating ROS-scavenging enzymes, such as peroxidases, catalase (CAT), superoxide dismutase to mitigate the oxidative effects (Bansal and Srivastava, 2012). Besides, multiple adaptive signaling pathways are triggered to avoid the negative impacts of flooding stress, such as phytohormone-signaling (Voesenek et al., 2003), Ca²⁺ cascade (Wilkins et al., 2016) and MAPK cascade (Singh and Sinha, 2016). These signaling pathways activate downstream transcription factors, and thereby modulate the regulation of stress-responsive genes and providing protective mechanisms (Hoang et al., 2017).

With the recent decrease in the cost of next-generation sequencing, the RNAseq-based transcriptome analysis has become one of the most efficient strategies in dissecting the genetic basis of complex traits variation, particularly the interaction between plants and abiotic stress (Zhang and Song, 2017). RNA-seq data has been demonstrated to provide global view of gene networks involved in flooding tolerance in maize (Du et al., 2017). In this context, RNA-seq has been performed in different crop species for understanding molecular mechanisms and signaling response underlying flooding tolerance in different crops, such as Arabidopsis (Van Veen et al., 2016), rice (Vijayan et al., 2018), and pea (Zaman et al., 2018). However, transcriptional responses to flooding stress have also been carried out in soybean, genes related to cell wall loosening, ethylene biosynthesis, alcohol fermentation, and pathogen defense were significantly up-regulated in soybean (Komatsu et al., 2009). Subsequent studies have identified genes associated with cell wall precursors and starch/sugar content, serving as an adaptive mechanism (Chen et al., 2016), as well as explored initial flooding tolerance mechanisms in soybean in response to flooding (Yin et al., 2017).

Annual wild soybean (Glycine soja Sieb. & Zucc.) being the progenitor of cultivated soybean (Glycine max (L.) Merr.) has been demonstrated to possess high genetic diversity compared to cultivated soybean (Nichols et al., 2007; Lee et al., 2008). Many studies have reported that wild soybean is not only rich in oil and protein content (Sebolt et al., 2000), but also revealed its extremely higher adaptability and resistance/tolerance to various biotic and abiotic stresses (Winter et al., 2007; Zhu et al., 2012), indicating the importance of wild soybean as a source of genetic variability. Thus, we can improve the seed-flooding tolerance of cultivated soybean by introducing favorable alleles from wild soybean into elite cultivars. Recent study has revealed that wild soybean accessions were more flooding tolerant relative to cultivated soybean (Valliyodan et al., 2016). Furthermore, the recent study carried out in our lab confirmed that the wild soybean accession PI342618B possess extremely high seed-flooding tolerance, and most the seedlings survived after 140 h of continuous seed-flooding treatment (Liu et al., 2013). However, till date, the molecular mechanisms and genetic basis underlying seed-flooding tolerance in soybean remain largely unexplored. Therefore, by keeping the above into view the present study has used RNA-seq approach to investigate transcriptomic profiles in the root tissues of two soybean species viz., PI342618B (SFT-tolerant) and NN86-4 (SFS-sensitive) subjected to seed-flooding stress at germination stage. The RNA-Seq approach provided an in-depth scenario on the whole soybean transcriptome and identified core genes associated with seed-flooding tolerance.