. 2016 Feb 9;17:102. doi: 10.1186/s12864-016-2420-0

Table 1.

Genes, proteins, metabolites, and promoters that are promising tools for the improvement of soybean drought responses

	Tissue	Observations	Comments
		Up-regulated genes
Homeobox leucine zipper	Leaves and roots	In leaf about 100-fold induced after 2 h and 150-fold after five hours. In root about 20-fold after five hours. The two proteins are 90.2 % similar.	The most similar Arabidopsis orthologue is ATHB12. The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities.
GLYMA16g02390
GLYMA07g05800
ERF/AP2	Leaves and roots	The most strongly up-regulated transcription factor gene in leaves after two hours with an inducition of 135-fold. Induced 36-fold after one hour in roots.	The most similar Arabidopsis gene is the ERF transcription factor ABA REPRESSOR1 (ABR1). ABR1 is expressed in response to ABA, osmotic stress, sugar stress and drought. Mutants are hypersensitive to these stresses.
GLYMA05g32040	Leaves and roots
ERF/AP2	Leaves and roots	The most strongly up-regulated transcription factor gene (573-fold after five hours in leaves). 10-fold induced in roots.	Another AP2/ERF transcription factor that is similar to Arabidopsis ABR1.
GLYMA20g30840	Leaves and roots
ERF/AP2	Roots	The third most strongly induced soybean gene at early time points in root and the most strongly up-regulated AP2/ERF gene.	Our yeast 2-hybrid analyses show that GLYMA03g26310 interacts with the drought inducible WRKY transcription factor GmWRKY53. Similar to AtERF13 that is involved in regulating various biotic and abiotic stresses.
GLYMA03g26310	Roots
ERF/AP2	Leaves	10-fold and 23-fold induced in leaves after five hours.	Our yeast 2-hybrid analyses show that both proteins interact with the drought inducible WRKY transcription factor GmWRKY53 [40].
GLYMA10g33810
GLYMA20g33800 (paralogs with 90.5 % identity)
GLYMA20g33800 (paralogs with 90.5 % identity)			Member of the subfamily B-3 of ERF/AP2 transcription factors. B-3 includes ATERF-6 that acts as a central regulator of leaf growth under water-limiting conditions in Arabidopsis.
WRKY	Leaves	Rapid and transient induction in leaves. Maximum of 71-fold induction after 3 h.	Group IIe WRKY transcription factor.
GLYMA01g43130 (GmWRKY161)	Leaves		Group IIe WRKY transcription factor.
WRKY	Leaves	Transiently up-regulated in leaves with a maximum of 21-fold after two hours.	Similar to AtWRKY6 that is implicated in regulating senescence, defence responses, arsenate uptake, boron deficiency, and low phosphate responses.
GLYMA17g04710 (GmWRKY112)	Leaves
NAC	Leaves and roots	Strongly induced in leaves with a maximum of 148–fold after three hours. Also strongly induced in roots with a maximum of 70-fold after five hours.	Apparent orthologue of Arabidopsis RD26/ANAC071 RD26/ANAC071 is induced in response to desiccation. It is localized to the nucleus and acts as a transcriptional activator in ABA-mediated dehydration responses.
GLYMA12g22880	Leaves and roots
NAC	Leaves and roots	Similar to GLYMA12g22880. Strongly induced in leaves with a maximum of 121–fold after three hours. Also strongly induced in roots with a maximum of 47-fold after five hours.	Similar to Arabidopsis RD26/ANAC071 and ANAC055 both of which appear to regulate stress responses.
GLYMA12g35000	Leaves and roots
bHLH	Roots	The most strongly early up-regulated transcription factor in roots (50-fold after 30 min) and the second highest induced gene at this time point.	Similar to bHLH92 (At5g43650) that functions in plant responses to osmotic stresses.
GLYMA12g22880	Roots
MYB	Leaves and roots	R2R3-MYB transcription factor 74-fold induced after three hours in leaves. 10-fold induced after five hours in roots.	Many MYB transcription factors regulate stress responses but the role of GLYMA05g35050 in unknown.
GLYMA05g35050	Leaves and roots
C3H	Leaves and roots	Up-regulated in the root 80-fold after three hours and 15-fold in the leaves after three hours.	SOMNUS is a key negative regulator of seed germination that acts downstream of PHYTOCHROME INTERACTING FACTOR3-LIKE5 (PIL5). The role of SOMNUS outside of seed germination is unclear.
GLYMA06g44440 (SOMNUS)	Leaves and roots
ERF/AP2	Leaves	Large induction of 427-fold after three hours in leaves. Not significantly induced in roots.	Similar to the tobacco proteins NtERF211 and NtERF204 both of which are strongly induced by drought.
GLYMA10g36760	Leaves
JAZ/TIFY	Leaves and roots	Rapidly up-regulated in roots with a later maximum of 61-fold induction after three hours. 18-fold induced in leaves after three hours.	TIFY5A-like transcription factor. Consistent with a role of JA in drought responses in soybean.
GLYMA15g09980	Leaves and roots
LEA protein	Leaves and roots	The most strongly up-regulated gene in the soybean genome (1,018-fold in leaves after three hours). Also up-regulated to a lower level in roots.	LEA proteins are well known drought response genes. The massive induction of this particular LEA gene suggests it could be a useful target for soybean improvement.
GLYMA10g07410	Leaves and roots
Glucose and ribitol dehydrogenase	Leaves and roots	One of only three genes induced over 1,000-fold by drought (1,009-fold after five hours in leaf). Also up-regulated to a lower level in roots.	May be involved in carbohydrate metabolism and the acquisition of desiccation tolerance (uniprot.org).
GLYMA03g39870	Leaves and roots
Auxin efflux carrier	Roots	610-fold induced in leaves.	Role in drought responses is unknown.
GLYMA11g09250	Roots	610-fold induced in leaves.	Role in drought responses is unknown.
Cytokinin hydroxylase-like	Leaves and roots	Very rapid early induction and maximum of 60-fold after five hours. 8-fold induction in leaves.	This suggests that trans-hydroxylation is involved in the regulation of cytokinin metabolism and signaling.
GLYMA10g37920			Potential for improving drought responses unknown.
Jasmonic acid carboxyl methyltransferase (JMT)	Roots	Root-specific early induction and maximum of 237-fold after five hours	Catalyzes the formation of methyl jasmonate from jasmonic acid and is a key enzyme for jasmonate-regulated plant responses (Seo et al., 200).
GLYMA16g24800	Roots
Lipoxygenase LOX1	Roots	Later root-specific induction with a maximum of 89 –fold after five hours.	Involved in the biosynthesis of JA
GLYMA13g42320	Roots		Involved in the biosynthesis of JA
ELI3-2 mannitol dehydrogenase	Roots	Strongly induced in both tissues with a maximum of 618-fold in leaves after five hours and 157 – fold in roots at the same time point.	Mannitol dehydrogenase (MTD) is a prime modulator of mannitol accumulation in plants (Zamski et al., 2001).
GLYMA01g02580	Roots
ABA2 (ABA deficient 2)	Leaves and roots	156-fold up-regulated in leaves after five hours and 32-fold induced in roots at the same time point.	ABA2 is an abscisic acid biosynthesis enzyme that belongs to a family of short-chain dehydrogenases/reductases. It is also called xanthoxin dehydrogenase. ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde. Abscisic aldehyde is then converted to ABA. ABA2 is a good candidate for improvement of soybean drought responses.
GLYMA11g18570	Leaves and roots
Protein phosphatase 2C (similar to AIP1, HIGHLY ABA-INDUCED PP2C GENE 2, HONSU)	Leaves and roots	72-fold up-regulated in leaveas and 31-fold in roots.	Similar to the HIGHLY ABA-INDUCED PP2C GENE 2 of Arabidopsis that functions as positive regulator of ABA (Lim et al., 2012).
GLYMA01g43460	Leaves and roots	72-fold up-regulated in leaveas and 31-fold in roots.
		Down-regulated genes
Stomatal Density and Distribution 1 (SDD1)	Leaves	mRNA level goes down 17-fold in leaves at the earliest time point. At this time point the eighth most strongly down-regulated gene.	Appears to be part of a long term response to drought that reduces the amount of stomata in new leaves.
GLYMA19g35200	Leaves
bHLH (Group 10 Ia)	Leaves	mRNA level goes down 31-fold in leaves after two hours. At this time point the tenth most strongly down-regulated gene.	Group 10 IA bHLH gene related to the regulators of stomatal development in Arabidopsis, FAMA, SPEECHLESS, and MUTE
GLYMA06g35330	Leaves
GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1)	Leaves	Within 30 min, the levels of mRNA encoding Glyma15g13840 and Glyma09g02881 fall to about one third of their non-stressed levels and reach a 7-9-fold reduction after 3–5 h.	GHR1 mediates ABA and hydrogen peroxide-regulated stomatal movement under drought stress [21] and GHR1 is a critical early component in ABA signaling.
GLYMA15g13840 and GLYMA09g02881	Leaves
		Constitutive genes
GmICHG	Roots	Among the 170 most highly expressed genes in soybean roots	The isoflavone conjugate-hydrolyzing β-glucosidase (GmICHG) may release conjugated coumestrol from its latent form in the vacuole to be excreted from the roots to promote plant-microbe interactions.
GLYMA12g05770	Roots	Among the 170 most highly expressed genes in soybean roots
		Metabolites
Coumestrol	Roots	An isoflavanoid with a striking 161-fold increase after three hours in roots. Levels have increased 46-fold after just one hour.	Previous reports show that coumestrol stimulates mycorrhizal colonization and hyphal growth and under certain conditions mycorrhizal plants can have improved drought tolerance. Possible novel drought tolerance mechanism where drought induces an increase in coumestrol, increased interactions with mycorrhiza and thereby enhances tolerance to drought stress. Coumestrol is therefore a potential biomarker for water stress and a promising target for legume improvement.
Formononetin	Roots	An isoflavanoid that increases almost 10-fold in roots.	Like coumestrol, formononetin may be involved in signaling to the rhizosphere as a response to drought.
Allantoin	Leaves and roots	Allantoin levels increase nearly 8-fold in leaves and 4.2-fold in roots.	The purine metabolite allantoin enhances abiotic stress tolerance through synergistic activation of abscisic acid metabolism [17]. Mutants that accumulate more allantoin show enhanced tolerance to drought.
Raffinose	Leaves and roots	In the roots there is a large increase in raffinose after three and five hours, reaching a peak of 12.89-fold increase after five hours of drought. A similar rise in leaves reaches 21.8-fold after five hours.	The raffinose pathway can provide osmolytes in situations of low water potential.
Galactinol	Leaves	In the leaf, galactinol increases 9.6-fold but there is no significant increase in roots.	Galactinol acts as an osmolyte in situations of low water potential.
γ-aminobutyrate (GABA)	Roots	GABA levels increase 13-fold in roots.	The GABA shunt is a stress response pathway, the functions of which include controlling cytoplasmic pH, maintaining C/N balance by converting glutamate in the cytosol to succinate in the TCA cycle, and aiding in oxidative stress protection by generating NADH and succinate.
		Hormones
ABA (abscisate/abscisic acid)	Leaves and roots	The ABA concentration increased 7.8-fold after five hours in leaf tissue and appears to increase over 5-fold in roots. Strong ABA2 up-regulation is consistent with increasing ABA levels. Many ABA responsive genes are up-regulated in both tissues. Components of ABA signaling such as protein phosphatase 2C genes are also up-regulated.	ABA plays a central role in regulating drought responses in soybean.
JA (Jasmonate)	Roots	There was a rapid rise in JA and its biologically active conjugate JA-ILE in the roots All of the biosynthetic enzyme genes in the JA biosynthetic pathway are rapidly and coordinately up-regulated in roots. Many JA signaling components such as JAZ repressors are differentially regulated.	JA clearly plays an important role in the response to drought in soybean. Lipoxygenase, allene oxidase synthase, allene oxidase cyclase, and 12-Oxo-PDA-reductase genes all show induction in roots and may be good targets for improvement of soybean.
Ethylene (Ethene)	Leaves and roots	Cyanoalanine (an indicator of ethylene biosynthesis) was elevated at the earliest time-point in leaf tissue suggesting that ethylene plays an early role in the response. The biosynthetic enzyme genes in the ethylene biosynthetic pathway show up-regulation.	Ethylene plays a role in the regulation of drought responses.
		Proteins
MAP kinase 2-like	Leaves	Increases 3.63-fold at the protein level.	Similar proteins in Medicago truncatula and Arabidopsis respond to many different stress stimuli.
GLYMA05g37480	Leaves	Increases 3.63-fold at the protein level.
Inositol-polyphosphate 5-phosphatase	Leaves	Increases 3.63-fold at the protein level.	A similar Arabidopsis protein (AT1G05630) is induced in response to ABA and wounding treatments.
GLYMA07g40360	Leaves	Increases 3.63-fold at the protein level.
		Promoters
ABRE CACGT/CG	Leaves and roots	The well-characterized ABA Response Element is found in the promoters of many of the most strongly up-regulated genes and the ABREs are clustered in the first 250–500 bp of the promoters.	The ABRE is a binding site for certain members of the bHLH and bZIP transcription factor families. Synthetic promoters containing ABREs or ABREs in combination with other drought responsive elements may prove useful for driving transgenes in projects aimed at improving drought responses.
CRT/DRE motif CAC/TCGACC	Leaves and roots	Found in ten of the root early up-regulated promoters	The Cold/Dehydration Responsive Element is the binding site for AP2/ERF transcription factors. Given that many ERF genes are strongly up-regulated by drought and that several are listed in this table as potential targets for improving drought responses then their potential binding sites are excellent candidates for building blocks for synthetic drought-inducible promoters.
GTGCnTGC/G Element	Leaves	Found by MEME in found in sixteen of the leaf late up-regulated promoters	Novel potential element. Will require detailed functional characterization and identification of cognate transcription factors.
GmWRKY71 and GmWRKY67 promoters	Roots	Drought inducible. The GmWRKY17 promoter is also responsive to ABA.	Drought and cold inducible promoters.
GmWRKY53 and GmWRKY112 promoters	Roots and leaves	Drought inducible. GmWRKY53 and −112 promoters respond positively to water stress through exogenous application of salt and PEG.	Drought and salt inducible promoters.