Discussion
Drought stress is a leading constraint affecting grain yield and
quality in soybean (Fried et al. , 2019). Genetic basis and
mechanisms underlying drought tolerance at germination stage has not
been extensively studied in soybean. Tolerance to drought stress is very
complex; hence, elucidation of molecular mechanism regulating drought
tolerance has been the long-term interest of soybean breeders.
Identification, cloning and exploitation of stress responsive genes
using molecular breeding/transgenic techniques is essential to develop
drought tolerant soybean cultivars. Although, linkage and association
mapping has identified genomic locus underlying drought tolerance
(Semagn et al. , 2013; Wang and Qin, 2017), but attempts to
understand drought tolerance at the transcriptome level was limited
especially at germination stage. In this context, the present study used
RNA-seq analysis to explore the global transcriptome of four contrasting
genotypes viz., two tolerant (DTP & DTL) and sensitive (DSP & DSL)
genotypes under drought stress at germination stage. Our results
revealed that tolerant genotypes (DTP & DTL) possess higher RL compared
to sensitive genotypes under stress treatment. This can be explained by
the fact that RSA are important sensors of drought tolerance, and
increased root length and deep root system greatly increases the
moisture absorption and nutrient extraction for plant survival under
water deficit stress (Wasaya et al. , 2018).
Both Venn diagram and Circos analysis suggests that sensitive genotypes
are comparatively more vulnerable to drought stress at the
transcriptomic level (Fig 2A, B & C ). Similar findings are
previously reported in several other crop plants under water-deficit and
other stresses ( Muthusamy et al. , 2016; Fracasso et al. ,
2016). These results can be explained by the fact that compared to
tolerant genotypes, sensitive genotypes reveal dramatic changes in
morpho-physiological and biochemical parameters while mitigating
negative impact of drought stress (Yang et al. , 2017). The higher
correlation of DTP with DTL compared to DSP and DSL provides explanation
about increased drought tolerance in DTL, because genes/alleles for
drought tolerance in DTL are derived from wild-parent/DTP, while same
genes/alleles for DSL comes from DSP (Fig. 3 ).
Both GO and MapMan analyses indicated that drought stress response was
highly enriched in the DEGs associated with water transport, cell wall,
plant-type vacuole membrane, antioxidant activity, catalytic activity,
auxin transport, peroxidase activity, protein kinase activity, TF
activity, secondary metabolite synthesis, hormone signaling and
signaling (Table 4 ). Under water deficit conditions, plant
cells must maintain functional integrity and rapidly remodeled to keep
cell wall flexible under abiotic stress (Houston et al. , 2016).
In this context, cell-wall remodeling enzymes such as pectin esterases
(PME), expansins, xyloglucan endotransglucosylase/hydrolase (XTH) as
well as glycine-rich cell wall structural protein (GRP) are involved in
maintaining cell wall rigidity, and permits to cope with water deficit
conditions (Tenhaken, 2015). However, most of these cell wall remodeling
enzymes were highly expressed in tolerant genotypes but down-regulated
in sensitive ones, indicating their vital role in cell wall remodeling
under drought stress (Table 4 ). For instance, Arabidopsis lines
overexpressing PMEI1 (pectin methylesterases) exhibited improved
germination rate and seedling root growth under water deficit condition
(An et al. 2008). Cho et al. , (2006) described that CaXTH3overexpressing-plants in Arabidopsis showed an increase in
drought tolerance, and XTHs have been reported to be
differentially regulated in maize under drought stress (Zhu et
al. , 2007). Furthermore, overexpression of expansin gene
(TaEXPB23 ) improves drought tolerance in tobacco. Similarly,AtEXP2 is involved in seed germination and drought stress
response in Arabidopsis (Yan et al. , 2014). Yang et
al. , (2014) also reported that overexpression of AtGRP2and AtGRP7 significantly influences drought tolerance in
transgenic rice, and revealed that rice plants overexpressingGRPs were more tolerant to water deficit relative to wild-type
plants. Xuan et al. , (2010) also demonstrated role ofNtGRP-1a transcripts under drought stress in tobacco. Hence,
up-regulation of above cell wall related genes might play essential role
in seed germination and drought tolerance.
Under stress conditions different secondary metabolites i.e., lipids,
amino acid and carbohydrate are accumulated in higher plants (Akula and
Ravishankar, 2011). Shikimate
pathway not only act as connection between central and secondary
metabolism but also serve as
precursor for most of the other secondary metabolites (Fig. S1 ;
Maeda and Dudareva, 2012). Biosynthesis of tyrosine and phenylalanine
through the shikimate pathway leading to the synthesis of wide range of
secondary metabolites (Less and Galili, 2008; Gill and Tuteja, 2010).
Isoquinoline alkaloids are derived from tyrosine, while
indole alkaloids are produced by metabolic engineering of the tryptophan
preventing plants from oxidative stress (Figure. S1 ; Sato and
Kumagai, 2013). Phenylalanine acts as a precursor for diverse secondary
metabolites and phenylalanine ammonia-lyase (PAL) takes part in
phenylpropanoid biosynthesis; an essential step towards biosynthesis of
anthocyanin’s, flavonoids, stilbenes, lignins and other compounds (Deng
and Lu, 2017). It is reported that lignin and anthocyanin’s biosynthesis
related genes were highly expressed under water stress in rice roots
(Yang et al. , 2006). In our findings, many unique genes related
to biosynthesis of lignin, alkaloids, flavonoids and anthocyanin’s such
as PAL, STR, and laccase were highly expressed in tolerant genotypes,
but mostly down-regulated in sensitive genotypes under drought
(Table 4 ), proposing their diverse role towards drought
tolerance. Among all the DEGs involved in the secondary metabolite
biosynthesis, highest number of genes associated with lignin
biosynthesis especially the laccase (LAC) and cinnamoyl-CoA reductase
(CCR) genes indicating essential role of lignin in drought tolerance.
Lignin reduces cell wall water penetration and transpiration in plants,
and also maintain cell osmotic balance and membrane integrity under
drought stress (Liu et al ., 2018). Moreover, laccase genes
participate in the oxidation of flavonoids, and plays fundamental role
in plant defense responses (Turlapati et al. , 2011). Flavonoids
serve as antioxidant, and provide shield to plants against abiotic
stresses (Pourcel et al. , 2007). For example, AtLAC2 inArabidopsis is involved in drought stress tolerance (Cai et
al. , 2006). Liu et al. , (2017) reported accumulation of
terpenoid indole alkaloids (TIAs) in the C. roseus under drought
stress. Hence, considerable upregulation of DEGs related to secondary
metabolite biosynthesis in drought-tolerant genotypes, suggesting their
vital role in drought tolerance.
Drought stress enhances reactive oxygen species (ROS) accumulation
resulting in cell wall degradation and membrane damage.(Helena and
Carvalho, 2008). Studies have demonstrated that ROS accumulation
impaired seed germination in different crops including soybean
(Ishibashi et al. , 2013). In this regard, plant utilize
antioxidant defense system for scavenging ROS to avoid oxidative stress,
and allows proper seed germination (Xie et al. , 2019). In our
data, many redox related DEGs including GST, POD, SOD, glutaredoxin and
thioredoxin were highly expressed in the drought-tolerant genotypes (DTP
and DTL), whereas the same genes were down-regulated in
drought-sensitive genotypes viz., DSP and DSL (Table 4 ). Many
studies have revealed induced expression of GST under various abiotic
stresses, including drought (Kumar and Trivedi, 2018). For instance,
Bhardwaj and Yadav, (2012) reported an increase in expression of GST,
POD, CAT, and SOD in horsegram subjected to drought stress, indicating
their important role in drought stress tolerance. In addition,
over-expression of AgAPX1 (ascorbate peroxidase) in Arabidopsis
led to increase in ascorbate content and drought tolerance (Liu et
al. , 2019). A glutaredoxin gene viz., SlGRX1 exhibited tolerance
to oxidative and drought stresses in tomato (Guo et al. , 2010).
Similarly, NADPH-dependent thioredoxin reductase A (NTRA ) mutant
exhibited tolerance to drought and oxidative stress (Cha et al .
2014).
In soybean and other crop species, many transcriptome studies has
revealed involvement of several TFs including, MYB, ERF/DREB, bHLH, NAC
and WRKY in seed-germination process under stress conditions (Leeet al. , 2015; Raineri et al. , 2016; Baillo et al. ,
2019). Members of these TF families may have either positive and
negative regulatory role in the drought stress tolerance. The MYB are
most abundant among all TFs expressed in present study, and many of
these TFs are differentially regulated in both tolerant and sensitive
genotypes (Table 3 ), suggesting their essential roles in
germination in response to drought stress. For example, OsMYB6overexpressing plants in rice enhanced tolerance to drought stress (Tanget al. , 2019). Similarly, Zhao et al. , (2018) reported the
involvement of TaMYB towards drought tolerance
in Arabidopsis . Overexpression of the ZmMYB3R andScMYBAS1 has been reported to enhance drought stress tolerance in
maize (Wu et al. , 2019) and rice (Peixoto-junior et al. ,
2018), respectively. After MYB, the bHLH followed by ERF, NAC and WRKY
represent the highest number of differentially expressed TFs under
drought stress. For example, bHLH family member ZmPTF1 andVvbHLH1 regulates drought tolerance in maize (Li et al. ,
2019) and Arabidopsis thaliana (Wang, et al. , 2016),
respectively by promoting root development, ABA synthesis and
accumulation of flavonoids. The AP2/ERF TF were revealed to modulate
brassinosteroid-regulated plant development and drought responses inArabidopsis (Xie et al. , 2019). Moreover, NAC genes such
as JUNGBRUNNEN1 in tomato (Thirumalaikumar et al. , 2018)
and OoNAC72 Arabidopsis (Guan et al. , 2019), positively
regulates drought tolerance, whereas, SbNAC052 , SbNAC073 ,
and SbNAC116 serve as negative regulator in drought stress
tolerance in sorghum (Sanjari et al. , 2019). Many TFs from WRKY
family exhibited function in drought tolerance in various crops, for
example overexpression of GmWRKY12 in soybean (Shi et
al.2018 ), TaWRKY2 and AtWRKY30 in wheat (Gao et
al. , 2018; El-Esawi et al. 2019), and ZmWRKY40 in Arabidopsis
(Wang et al.2018 ) has led to enhanced drought tolerance in these
crop plants. Hence, these TFs can be an important target for breeding
drought tolerance in soybean.
In the present study, many genes related to plant-specific
Ca2+ signaling such as calcium-binding proteins,
calcium ATPases, calmodulin-like proteins (CMLs), calmodulin-binding
protein, calmodulin-binding receptor, Ca2+-dependent
protein kinases (CPKs), and Annexin were mostly up-regulated in tolerant
genotypes as compared to sensitive genotypes. (Table 4 ). For
example, Campo et al. , (2014) reported that OsCPK4overexpression in rice showed an increase in drought tolerance by
reducing lipid peroxidation and electrolyte leakage. Plasma membrane
Ca2+-ATPase directly regulates drought stress
tolerance by activating ABA signaling pathway (Shao et al. , 2008;
Cerana et al. , 2006) and the increased ABA accumulation leads to
stomatal closure and expression of many stress-related genes. For
example, Cerana et al ., (2006) reported overexpression
of Ca2+-ATPase viz.,ACA8 and ACA9 stimulated ABA accumulation. Hence, it has
been proposed that Ca2+-ATPases might play important
role in drought stress response through ABA signaling, as the latter is
well-established mediator of drought stress adaptation in plants
(Qudeimat et al. , 2008). Calmodulin-like Proteins such asCML20 in Arabidopsis (Wu et al. , 2017),ShCML44 from Solanum habrochaites (Munir et al. ,
2016) were revealed to induce drought tolerance by regulating ABA
signaling in guard cells. The annexin gene AnnSp2 exhibited
drought tolerance in overexpressed transgenic tomato plants through
ROS-scavenging and modulation of ABA synthesis (Ijaz et al. ,
2017). Wei et al., (2014) reported that OsCPK9,calcium-dependent protein kinase in rice is involved in tolerance to
drought stress. Hence, the above findings suggest close relationship
between ABA and Ca-signaling in drought stress response. Evidence
indicate that Ca2+/CaM is involved in ABA-induced
drought signaling under PEG stress, and ABA-synthesis was associated
with cytoplasmic Ca2+ concentrations (Li et al .
2002). As reported previously, ABA activates cytosolic
Ca2+ in guard cells by maintaining turgor within guard
cells that leads to stomatal closer and prevention of transpiration
water loss, and ultimately induces drought tolerance (Song et
al. , 2008). Furthermore, receptor-like protein kinase (RLKs) and
mitogen-activated protein kinases (MAPKs) are key components for
signaling pathways in plant and have diverse function in seed
germination by regulating stress-responsive gene ( Baek et al. ,
2019; Jagodzik et al. , 2018). For instance, seed germination was
significantly enhanced by expression of SpMAPK3 andSlMAPK3 in response to abiotic stresses (Muhammad et al. ,
2019; Li et al. , 2014). The MAPK kinase10.2 promotes
drought tolerance by activating different MAPKs in rice (Maet al. , 2017). Cysteine-rich RLKs such as CRK45 inArabidopsis (Zhang et al. , 2013) and TaCRK41 in
wheat (Chen et al. , 2017) involved in ABA signaling and
positively regulates seeds germination under drought and oxidative
stress. Similarly, in Arabidopsis, proline-rich RLKs, PnLRRRLK27exhibited tolerance towards abiotic stress during seed germination (Wanget al. , 2017). Consistent with these results, we also
observed up-regulation of MAPKs and RLKs in tolerant genotypes, whereas,
reverse trend was seen in sensitive genotypes. Therefore, the above
results propose that MAPKs, RLKs and Ca2+ signaling
together with ABA might play key role to regulate seed germination under
water deficit conditions.
Numerous transgenic studies have demonstrated that overexpression of
Aquaporins (AQPs) viz., TIPs and PIPs enhanced drought tolerance
probably by endorsing stomatal closure and regulating the plant
hydraulics (Zargar et al. , 2017). For example, VfPIP1overexpressing plants in Arabidopsis thaliana exhibits drought
tolerance through promoting stomatal closure (Cui et al. , 2008).
Pou et al. , (2013) reported the putative role of PIP andTIP genes in leaf hydraulic and stomatal conductance in grapevine
under drought stress. Although role of AQPs in dehydration tolerance has
been extensively studied in plants but still conflict of interest
remained among the researchers regarding their up- or down-regulation
during water deficit condition. Under drought stress, considerable
variation was observed in the expression of PIPs at the
transcript level; for example, significant upregulation of somePIPs was observed while expression of others remained either
unchanged or downregulated (Lian et al. , 2006; Guo et al. ,
2006). Upregulation of some AQPs might assist in maintaining the normal
physiological processes in plant and resist the stress, while other may
help to adapt or tolerate the stress condition by reducing their own
activity and expression (Zargar et al . 2017). Hence, in the
present study some unique AQP genes are differentially regulated in
tolerant and sensitive genotypes proposing their innate role in drought
tolerance (Table 4 ).
Phytohormone signaling is complex and plays important regulatory role in
drought responsive pathways of soybean (Pandey et al , 2017). In
recent study, genes associated with phytohormone biosynthesis and
signaling viz., AUX, ETH, ABA and GA represent most number of DEGs in
both tolerant and sensitive genotypes under drought stress
(Table 2; Table 4 ). For example, auxin/indole-3-acetic acid
(Aux/IAA), small auxin‐up RNA (SAUR), indole-3-acetic acid-amido
synthetase/Gretchen Hagen (GH3) and auxin efflux carrier component (PIN)
showed dynamic changes in the roots of tolerant and sensitive genotypes
under drought stress; however, these genes are highly expressed in
tolerant genotypes, whereas, reverse trend was observed in sensitive
genotypes, indicating complex role of auxin signaling. Zhang et
al. (2012) revealed that auxin efflux carrier component such asOsPIN3t in rice involved in auxin transport and response to water
stress. The auxin-sensitive Aux/IAA proteins has been revealed to
mediate drought tolerance through regulating glucosinolate levels in
Arabidopsis (Salehin et al. , 2019). Under drought stress,
overexpression of auxin-responsive protein, TaSAUR75 exhibited an
increase in root length and survival rate
in Arabidopsis(Guo et al. , 2018). Similarly, GH3 genes of Chickpea
(CaGH3-1 & CaGH3-7 ) and Medicago truncatula(MtGH3-7 , MtGH3-8 and MtGH3-9 ) were highly
induced under drought stresses (Singh et al. , 2015). In our
findings, many GH3 genes were highly induced in tolerant
genotypes, respectively, suggesting their important role in stress
adaptation. Ethylene biosynthesis mainly involved two enzymes viz.,
aminocyclopropane-1-carboxylic acid (ACC) oxidase and ACC-synthase (ACS)
in various crops (Yoon and Kieber, 2013; Van Der Straeten et al .,
2001), and this hormone regulates seed germination and seedling growth
under abiotic stresses (Huang et al. , 2019). Similar with these
findings, our study also exhibited higher expression levels of ACS and
ACO in tolerant genotypes. In addition, it is also reported that
hormonal balance between ABA and GAs is necessary to regulate seed
germination (Miransari and Smith, 2014). In present study, DEGs
associated with gibberellin 2-beta-dioxygenase, Gibberellin
3-beta-dioxygenase, Gibberellin 20 oxidase, and DELLA protein were
highly expressed in tolerant genotypes as compared to sensitive
genotypes (Table 1). Habib et al. , (2019) demonstrated
that SlGRAS7 TF improved drought tolerance by enhancing
gibberellin/auxin signaling. Consistent with a recent report, we also
identified many enzymes related to ABA biosynthesis such as
9‐cis‐epoxycarotenoid dioxygenase (NCED), abscisic acid 8′-hydroxylase
and carotenoid cleavage dioxygenases (CCD). These enzymes were
differentially regulated in tolerant and sensitive genotypes resulting
in better hormonal balance under drought stress (Shu et al. ,
2018). In plants, ABA plays diverse role in response to drought as well
as in various developmental processes such as seed germination. Drought
stress enhances plant ABA accumulation resulting in ABA-receptor complex
(PYR/PYL/RCAR–PP2C–ABA) that triggers SnRK2 protein kinases, and this
kinase facilitate stomatal closure and reducing transpiration water
loss, and maintaining water balance within plant under water-deficit
condition (Fujita et al. , 2013). Brassinosteroids (BRs) are plant
steroid hormones, which regulate the expression of stress related genes.
Brassinosteroid Insensitive 1 (BRI1) was highly expressed in tolerant
genotype, and previous study has revealed that BRI1 plays
essential role in plant growth, development and drought tolerance. For
example, Feng et al. , (2015) using RNAi suggested that mutation
of bdBRI1 in Brachypodium distachyon resulting in dwarf
phenotype with enhanced tolerance towards drought stress.
Based on the findings of GO enrichment, PPI network and dChiP analysis
as well as screening of genes within previously reported genomic/hotspot
regions associated with drought tolerance in soybean on Chr.08, a total
of ten genes were considered as the most possible candidate genes
regulating drought tolerance at germination stage in soybean
(Table 5 ). Function annotation revealed thatGlyma08G162700 function as peroxidase 3 (POD 3),Glyma08G091400 as ‘glutamate decarboxylase 1-like’,Glyma08G097300 as peroxisomal (S)-2-hydroxy-acid oxidase
GLO1-like , Glyma08G045000 as Ca-transporting ATPase,Glyma08G271600 function as glycine-rich cell wall structural
protein, Glyma08G014200 function as tubulin beta-1 chain-like,Glyma.08G201700 function is uncharacterized; the remaining three
predicted genes viz., Glyma08G042100 , Glyma.08G029400 andGlyma.10G184500 function as MYB184, MYB127 andMYB78, respectively (Table 5) . The POD 3 enzymes were
reported to participate in plant development, stress responses and
hormone signaling (Wu et al. , 2019); for example, AtPrx3participates in positive regulation drought stresses response in
Arabidopsis (Llorente et al. , 2002). Yong et al . (2017)
reported that inhibition of glutamate decarboxylase activity result in
the increase of endogenous glutamate (Glu), and that in turn enhanced
drought tolerance in white clover. Down-regulation of glycolate
oxidase pathway reduces peroxisomal
H2O2 production in the green tissues of
plants under drought stress, and provides tolerance to oxidative stress
(Zhou et al ., 2007; Noctor et al ., 2014). The
Ca2+ATPase such as OsACA6 confers drought
stress tolerance with reduced accumulation of ROS and enhanced the
expression of stress‐responsive genes in tobacco (Huda et al. ,
2013). Involvement of Ca2+-ATPase in drought tolerance
has been revealed through transcript profiling of a sweet potato (Yanget al. , 2018). Moreover, in B. napus , two cell-wall
related proteins viz., glycine-rich and fasciclin-like arabinogalactan
were induced under drought stress (Koh et al. , 2015). Panet al . (2018) reported highly induced expression of tubulin
beta-1 chain-like in foxtail millet under drought stress (Pan et
al. , 2018). The tubulin beta-1 (R83) chains are major constituents of
microtubules and their accumulation also peaked at 48 h after the onset
of drought stress (Bian et al. , 2017). In addition, many of the
earlier studies has explained the important role of MYB gene family in
the drought stress tolerance in crop plants (Tang et al ., 2019;
Zhao et al ., 2018; Wu et al ., 2019; Zhang et al .,
2019; Peixoto-Junior et al ., 2018). Hence, the above ten genes
that includes three hub genes were predicted as the most possible
candidate genes regulating drought tolerance at germination stage in
soybean. Therefore, these genes required further validation to prove
their actual role and use in soybean improvement.
Based on the above findings, a hypothetical model was proposed as shown
in Fig. 11 . This comparative model explains, how aquaporin’s,
cell-wall related enzymes, secondary metabolites, antioxidants, kinases,
MAPK signaling and TF activities functions in drought response between
drought tolerant and sensitive genotypes.