GmSAP5,a soybean A20/AN1 domain-containing stress-associated protein gene activated by GmAREB3,increases drought stress resistance in soybean by mediating ABA signaling

Zeho Hou,Xingzhn Zhng,2,Yimio Tng,Tifei Yu,Lei Zheng,Jun Chen,Yongbin Zhou,Yongwei Liu,Ming Chen,Zho-Shi Xu,*,Youzhi M

a Institute of Crop Sciences,Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement,Key Laboratory of Biology and Genetic Improvement of Triticeae Crops,Ministry of Agriculture,Beijing 100081,China

b Beijing Engineering Research Center for Hybrid Wheat,The Municipal Key Laboratory of the Molecular Genetics of Hybrid Wheat,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,China

c Institute of Genetics and Physiology,Hebei Academy of Agriculture and Forestry Sciences/Plant Genetic Engineering Center of Hebei Province,Shijiazhuang 050051,Hebei,China

Keywords:Soybean GmSAP5 GmAREB3 Drought stress ABA

ABSTRACT Drought stress impairs crop growth and productivity.Stress-associated proteins(SAPs),a class of zinc finger proteins containing the A20/AN1 domain,function in various stress responses in plants.However,little is known about the function of SAPs in drought-stress responses in soybean,an oil and protein crop.We report that a GmSAP5 protein confers drought tolerance by increasing sensitivity to abscisic acid(ABA)and reducing stomatal aperture.Overexpression and RNA interference of GmSAP5 in soybean hairy roots resulted in elevated resistance and sensitivity to drought stress,respectively.ABA and proline contents increased in GmSAP5-overexpressing plants under water-deficit conditions.Lower water loss rates and higher relative water contents were observed in GmSAP5-overexpressing plants,resulting in increased drought-stress resistance.A yeast one-hybrid assay and luciferase transient transcriptional activity assay showed that GmAREB3,an AREB/ABF transcription factor,could bind to the promoter of GmSAP5 and activate its expression.These results suggest that GmSAP5 acts downstream of GmAREB3 and improves drought-stress resistance by mediating ABA signaling.

1.Introduction

Drought stress severely impairs plant growth,productivity,and quality and threatens global food security[1,2].Plants have evolved various biochemical,physiological,and molecular mechanisms to adapt to,or avoid harmful environmental conditions[3].Numerous abiotic stress-related transcripts and proteins have been identified by transcriptomics and proteomics,and these transcripts and proteins are generally divided[2]into two major groups.The first group is involved in membrane protection and includes reactive oxygen species(ROS)detoxification enzymes,antioxidants,and osmoprotectants[4],whereas the second group is composed of transcription factors(TFs)or regulatory proteins,which are involved in signaling cascades and transcriptional control[5,6].These drought-responsive genes have been recruited to form a complex response network that protects and maintains cellular structures and increases drought tolerance[7-9].

Abscisic acid(ABA)is a phytohormone that functions in plant response to abiotic stress.ABA levels increase markedly under osmotic stress,stimulating stomatal closure,controlling transpiration,changing gene transcription,and activating adaptive biochemical and physiological responses[4,10].The expression of osmotic stress-responsive genes is regulated by ABA-dependent and ABA-independent signaling pathways[11,12].ABAresponsive elements(ABRE,ACGTGG/TC)in promoters and ABREbinding protein/ABRE-binding factors(AREB/ABFs)function in the regulation of drought stress-induced expression of genes in the ABA-dependent pathway[12,13].Numerous other stressresponsive genes,such as DRE-/CRT-binding protein 2(DREB2)TFs,RD29A,and COR15A,are induced via the ABA-independent pathway[14,15].For this reason,several stress-inducible genes involved in the ABA-dependent and ABA-independent signaling pathways have been investigated for their ability to improve plant drought-stress tolerance by genetic engineering[16,17].

Stress-associated proteins(SAPs),which are a class of zinc finger proteins containing the A20/AN1 domain,have emerged as candidates for biotechnological improvement of plant tolerance to a variety of abiotic and biotic stresses[18-20].OsSAP1,the first A20/AN1 zinc finger domain SAP gene identified in rice,improves cold,drought,and salt tolerance in transgenic tobacco without any growth penalty[21].Another rice A20/AN1-type zinc finger protein,ZFP185,is involved in the gibberellin(GA)and ABA biosynthesis pathways,and overexpression of ZFP185 increases sensitivity to abiotic stress[22].Overexpression of Aeluropus littoralis AlSAP and OsSAP11 in transgenic plants has also been shown to protect yield against drought and salt stress[23-25].

Although analysis of SAP gene expression in many species has strengthened the association of the SAP gene family with abiotic stress response[18],little is known about the functions of SAP genes in drought-stress responses in soybean(Glycine max),an oil and protein crop worldwide that is vulnerable to water-deficit stress[26,27].We report that a stress-responsive SAP gene,GmSAP5,confers drought tolerance by increasing sensitivity to ABA and reducing the stomatal aperture.GmAREB3 binds directly to the promoter region of GmSAP5 and activates its expression.Based on these findings,we present a molecular model of the functions of GmAREB3 and GmSAP5 in soybean under drought stress conditions.

2.Materials and methods

2.1.Phylogenetic and domain analysis

The amino acid sequences of AtSAP5(accession ID:AT3G12630),OsSAP8(AAQ84334),OSISAP1(XP_015651267),ZFP185(XM_464458),OsSAP11(XP_015651039),ZFP177(AY282740),AtSAP10(AT4G25380),AtSAP12(AT3G28210),SbSAP14(XP_002466323),MusaSAP1(FF557701),MtSAP1(XP_003625187),TaSAP5(TraesCS5B02G262900),and AlSAP(ABK90631)were retrieved and used to construct a phylogenetic tree with MEGA-X software[28].Multiple sequence alignment of the amino acid sequences was performed with ClustalW software(v2.1),and online tools from Pfam(https://pfam.xfam.org/)were used for identifying conserved domains.

2.2.Subcellular localization assay

The coding sequence of GmSAP5 was isolate d from leaf cDNA of the soybean cultivar Zhonghuang 39 and inserted into the p16318-GFP vector to produce a GmSAP5-green fluorescent protein(GFP)fusion construct using ABclonal Multif seamless assembly kit(ABclonal,Beijing,China).The primers used are listed in Table S1.The p16318-GFP empty vector was used mainly as a control to produce free GFP.For co-localization studies,a nuclear localization signal(NLS)-protein[29]fused with red fluorescent protein mCherry was used as a nuclear marker.The recombinant plasmids were transiently expressed in A.thaliana leaf mesophyll protoplasts 18 h after PEG-calcium-mediated transformation[30],and a confocal laser-scanning microscope(LSM700,Carl Zeiss,Germany)was used to visualize the fluorescent signals of GmSAP5-GFP in subcellular organelles.

2.3.GmSAP5 promoter-GUS activity assay

The 2-kb promoter region upstream of the 5′untranslated region(5′UTR)of GmSAP5 was amplified from genomic DNA using specific primers(Table S1)and ligated into the pCAMBIA1305-GUS vector.The resulting vector,proGmSAP5::GUS,was transformed into the wild-type(WT)A.thaliana ecotype Col-0 background by Agrobacterium-mediated transformation[31].T0transgenic plants were selected using hygromycin and confirmed by PCR amplification.T3transgenic lines were used for histochemical β-glucuronidase(GUS)activity assays.Seven-day old proGm-SAP5::GUS lines were transferred to 1/2 Murashige and Skoog(MS)medium containing 9% PEG 6000(m/v)and 5μmol L-1ABA for stress treatment.After two days of treatment,the tissues were stained using a GUS staining kit(Real-Times,Beijing,China)according to the manufacturer’s protocol.The GUS-stained tissues were visualized under an Olympus DP73 microscope(Olympus,Japan).

2.4.Overexpression of GmSAP5 in A.thaliana and stress treatment

To produce GmSAP5 overexpression(OE)lines,the coding sequence of GmSAP5 was isolated and cloned into the pCAMBIA1302 vector and transferred into the Agrobacterium strain GV3101.The GmSAP5-OE vector was transformed into the A.thaliana ecotype Col-0 background by Agrobacterium-mediated transformation[31].Transgenic plants were selected using the herbicide hygromycin,and the presence of the transgene was verified by PCR amplification and quantitative reverse transcription PCR(qRT-PCR).

For germination assays,the seeds of the GmSAP5-OE lines and WT were placed on 1/2 MS medium with different concentrations of PEG 6000(0,6%,and 9%)or ABA(0,0.3μmol L-1,and 0.5μmol L-1),incubated at 4 °C in darkness for 3 days,and then grown at 23°C(with a 16 h/8 h light/dark photoperiod)to record the germination rate.For root-length assays,5-day-old seedlings were transferred from 1/2 MS medium plates to 1/2 MS medium containing different concentrations of PEG 6000(0,6%,and 9%)or 5μmol L-1ABA,and cultured vertically under normal conditions.The primary root length of seedlings from each line was calculated after treatment for 1 week.For drought-tolerance assays,3-week-old seedlings were transferred from 1/2 MS medium plates to soil in a single pot and acclimatized for 1 week.The seedlings were subjected to water-deficit stress for 25 days.

2.5.Overexpression and suppression of GmSAP5 in soybean hairy roots

The coding sequence of GmSAP5 was isolated and cloned into the pCAMBIA3301 vector containing a 35S promoter to generate a pCAMBIA3301-GmSAP5-OE vector.An 821-bp hairpin RNA fragment containing the coding sequence(positions 29 to 328)was synthesized and ligated into the vector pCAMBIA3301 to generate a pCAMBIA3301-GmSAP5-RNAi suppression vector.The pCAMBIA3301-GmSAP5-OE,pCAMBIA3301-GmSAP5-RNAi,and empty pCAMBIA3301 vectors were transformed into Agrobacterium strain K599 and then transformed into the hypocotyls of 5-day-old soybean seedlings as described previously[32].The transcription level of GmSAP5 in hairy roots was measured by qRT-PCR.The primers are listed in Table S1.Fifteen composite soybean plants containing non-transgenic leaves and transgenic hairy roots were investigated to quantify drought tolerance and evaluate changes in physiological parameters.

2.6.Measurement of physiological parameters

Changes in malondialdehyde(MDA)and proline concentration were assayed with MDA and proline assay kits(Suzhou Comin,Suzhou,China).ABA content was assayed with the Plant Hormone Abscisic Acid(ABA)ELISA Kit(Jianglai,Shanghai,China)according to the manufacturer’s instructions.Relative water content(RWC)[33]and water loss rate[34]were calculated as described previously.Stomatal aperture assays were performed as described previously[35].

2.7.Stress treatment in soybean plants and qRT-PCR analysis

Two-week old-soybean(Zhonghuang 39)seedlings were subjected to dehydration stress and phytohormone treatments.For dehydration stress,the seedlings were removed from soil and treated with 15% PEG 6000.For phytohormone treatments,soybean seedlings were transferred to Hoagland’s solution containing 100μmol L-1ABA.The seedlings were sampled at 1,2,4,8,12,and 24 h after treatment,and seedlings before stress treatment were collected as the control.Three biological replicates were tested,and the samples were immediately frozen in liquid nitrogen and stored at-80 °C until use.

Total RNA was isolated from each sample using the Plant Total RNA Kit(Beijing Zoman,Beijing,China),and first-stand cDNA was synthesized using the TransScript First-Strand cDNA Synthesis SuperMix(Transgen,Beijing,China)following the manufacturer’s protocol.qRT-PCR was performed on an Applied Biosystems 7500 real-time PCR system using TransStart Top Green qPCR SuperMix(Transgen,Beijing,China)according to the manufacturer’s instructions.The primers are listed in Table S1,and the 2-ΔΔCT method[22]was used to calculate relative transcript levels.

2.8.Yeast one-hybrid(Y1H)assay

A soybean leaf cDNA library was constructed using CloneMiner II cDNA Library Construction Kit(Invitrogen,Waltham,MA,USA)according to the manufacturer’s instructions and the cDNA library was cloned into the pGADT7 vector fused in-frame with the GAL4 activation domain to generate a library for yeast one-hybrid(Y1H)screening.The promoter of GmSAP5 was cloned into the pAbAi vector and the construct was linearized at BstBI sites and transformed into Y1HGold to generate reporter strains.Using the Y1H system(Takara,Shiga,Japan)to screen the soybean yeast cDNA library,we obtained four proteins(one GmAREB3,two RNA polymerases,and one ethylene-responsive transcription factor)able to bind to the GmSAP5 promoter.To reconfirm the Y1H screening results,the 2 kb promoter region upstream of the 5′UTR of GmSAP5 was amplified using specific primers and cloned into the XhoI site of the pLacZi vector to construct the pLacZi-ProGmSAP5recombinant vector.The coding sequence of GmABRE3 was cloned and ligated into the EcoRI sites of pB42AD to form the pB42AD-GmAREB3 expression vector.The primers are listed in Table S1.The recombinant plasmids were co-transformed into EGY48 yeast cells and cultured on SD-Trp/-Ura double deficiency medium.Single clones harboring different vector combinations were transferred to SDTrp/-Ura medium containing X-α-Gal and color changes in the yeast cells were observed after incubation at 30 °C[36].

2.9.Transient transactivation assay

Transient transactivation assays were conducted in A.thaliana protoplasts as previously described[37].The 2-kb genomic sequence of the GmSAP5 promoter was amplified and cloned into the HindIII sites of the pGreenII 0800-LUC vector as a reporter.The coding sequence of GmAREB3 was amplified and cloned into the NcoI and BstEII sites of the pCAMBIA1302 vector as an effector,and the empty pCAMBIA1302 vector containing the GFP coding sequence was used as a control.Transient transactivation assays were performed using A.thaliana leaf mesophyll protoplasts,and the renilla luciferase(REN)gene was used as the internal control.The E1910 Dual-Luciferase Reporter Assay System(Promega,Madison,WI,USA)was used to detect luciferase activity after the infected leaves were treated with or without 5μmol L-1ABA for 16-18 h.Three independent experiments were performed and 10 technical replicates were assayed.

Fig.1.Bioinformatics and expression pattern analysis of GmSAP5 and subcellular location of GmSAP5.(A)Phylogenetic relationship between GmSAP5 and other SAP proteins.The SAP-homologous proteins used are AtSAP5,OsSAP8,OSISAP1,ZFP185,OsSAP11,ZFP177,AtSAP10,AtSAP12,SbSAP14,MusaSAP1,MtSAP1,TaSAP5,and AlSAP.(B)Multiple amino acid sequence alignment of SAP proteins.(C)Expression of the GmSAP5 gene in various tissues determined by qRT-PCR analysis.(D)Subcellular localization of the GmSAP5-GFP protein in A.thaliana leaf mesophyll protoplasts.GmSAP5-GFP was co-expressed with a nuclear-marker(NLS-RFP).Scale bars,10μm.

2.10.Overexpression of GmAREB3 in soybean hairy roots

The coding sequence of GmAREB3 was isolated and cloned into the pCAMBIA3301 vector containing a 35S promoter to generate a pCAMBIA3301-GmAREB3-OE vector.The pCAMBIA3301-GmAREB3-OE and empty pCAMBIA3301 vectors were transformed into Agrobacterium strain K599 and then transformed into the hypocotyls of 5-day-old soybean seedlings as described previously[32].The transcription of GmAREB3 in hairy roots was measured by qRT-PCR analysis.The primers are listed in Table S1.Fifteen composite soybean plants containing non-transgenic leaves and transgenic hairy roots were investigated to quantify drought tolerance and to evaluate changes in physiological parameters.

3.Results

3.1.GmSAP5 is a cytoplasmic and nuclear-localized zinc finger A20/AN1 domain-containing stress-associated protein

Our previous comparative RNA-sequencing analysis revealed high expression of GmSAP5 under drought-stress conditions compared with normal conditions(Fig.S1).The coding sequence of GmSAP5 was cloned from soybean leaf cDNA.The GmSAP5 gene contains a 495-bp coding sequence encoding a 17.63-KDa protein.Phylogenetic analysis showed that the soybean GmSAP5 protein was closely related to SAP proteins reported as positive regulators of abiotic stress response in previous studies:AtSAP5[18],OSISAP1[20],TaSAP5[19]and OsSAP11[20](Fig.1A).Domain search showed that the GmSAP5 protein contains a zf-A20 domain and zf-AN1 domain(Fig.1B).GmSAP5 was expressed mainly in roots and stem(Fig.1C).And subcellular localization assay showed that the GmSAP5-GFP fusion protein was present in the cytoplasm and nucleus(Fig.1D).

3.2.Drought stress and ABA treatment induced GmSAP5 expression

GmSAP5 promoter-GUS transgenic lines were generated to further examine the expression pattern of GmSAP5.GUS staining was detected in leaf veins,nodes,hypocotyls,and root tips,and the expression of proGmSAP5::GUS was also increased by both PEG 6000 and ABA treatments(Fig.2A).qRT-PCR was performed to investigate the expression pattern of GmSAP5 in response to drought stress and ABA treatment.GmSAP5 transcript levels quickly accumulated at 1 h after PEG 6000 treatment,but showed a decrease at 4 h,and then steadily continued to increase,reaching a maximum(a greater than 20-fold induction)at 24 h under osmotic stress(Fig.2B).In response to ABA treatment,the transcript abundance of GmSAP5 was significantly induced but decreased markedly at 8 h(Fig.2C).Thus,the expression of GmSAP5 could be increased by osmotic stress and ABA treatment.

3.3.GmSAP5-overexpressing A.thaliana lines are sensitive to exogenous ABA

Fig.2.Drought stress and ABA treatment induce the expression of GmSAP5.(A)GUS assay of leaf,node,hypocotyl,and root of proGmSAP5::GUS transgenic plants,and assay of GUS activity following PEG 6000 and ABA treatments.Scale bars,1 cm in(a),0.25 cm in(b),(c),and(d),and 2 cm in(e),(f),and(g).(B,C)Expression patterns of the GmSAP5 gene under PEG 6000 and ABA treatments,determined by qRT-PCR analysis.Asterisks indicate significant differences by two-tailed Student’s t-test(*,P＜0.05).

Fig.3.GmSAP5-overexpressing plants show increased sensitivity to ABA.(A,B)Seed germination assay of wild-type(WT)and transgenic overexpression(OE)plants under control and exogenous ABA conditions.(C)Seedlings at 7 days after transfer to control 1/2 MS medium plates or plates containing 5μmol L-1 ABA.Seedlings were 5 days old at the time of transfer.Scale bars,2 cm.(D)Stomatal aperture analysis of wild-type and transgenic OE plants treated with exogenous ABA.Scale bars,10μm.(E,F)Measurement of root length and stomatal aperture calculated from(C)and(D).Asterisks indicate significant differences by two-tailed Student’s t-test(*,P＜0.05).

Fig.4.Overexpression of the GmSAP5 gene increases drought tolerance in terms of germination rate and primary root growth in Arabidopsis thaliana.(A-C)Seed germination assay of wild-type and transgenic overexpression(OE)plants under control,6%PEG 6000,and 9%PEG 6000 conditions.(D-F)Seedlings at 7 days after transfer to control 1/2 MS medium plates or plates containing 6% or 9% PEG 6000.Scale bars,2 cm.(G)Measurement of root length calculated from(D),(E),and(F).Asterisks indicate significant differences by two-tailed Student’s t-test(*,P＜0.05).

To investigate whether GmSAP5 is involved in ABA signaling,three independent GmSAP5-overexpressing lines(OE1,OE2,and OE6)were generated and the transcription of GmSAP5 was confirmed by qRT-PCR(Fig.S2).Next,we compared ABA sensitivity between the OE and WT plants during the germination and vegetative growth stages,and found that the germination rate of the OE plants was lower than that of the WT before 72 h under ABA treatment(Fig.3A,B).Primary root growth and stomatal opening of OE plants were significantly more inhibited by ABA than those of WT plants(Fig.3C-F).These results suggest that GmSAP5 plays a role in ABA signaling during the germination and vegetative growth stages.

3.4.Overexpression of GmSAP5 increases drought tolerance in terms of primary root growth in A.thaliana

We compared the drought tolerances of the OE and WT plants during the germination and vegetative growth stages.The germination rate of OE plants was markedly higher than that of WT plants grown in 1/2 MS medium containing 6% and 9% PEG 6000(Fig.4A-C).Compared with the WT plants,the primary root growth of GmSAP5 transgenic plants was markedly less inhibited by PEG 6000 treatment(Fig.4D-G).

3.5.Phenotypic characterization of GmSAP5-overexpressing A.thaliana plants under drought stress conditions

Fig.5.GmSAP5-overexpressing transgenic Arabidopsis plants show increased tolerance to drought stress compared with wild-type plants.(A)Wild-type(WT)and overexpression(OE)plants were exposed to control conditions or drought stress.(B,C)Water loss rate(B)and survival rate(C)of seedlings were calculated from the results of three independent experiments.(D-F)Changes in the malondialdehyde(MDA)content(D),proline content(E),and ABA content(F)of seedlings.(G-I)Expression profiles of AtGolS2(G),AtDREB2C(H),and AtP5CS1(I)under control and drought-stress conditions measured by qRT-PCR.Asterisks indicate significant differences by two-tailed Student’s t-test(*,P＜0.05).

The drought tolerance of GmSAP5-overexpressing lines during the seedling stage was investigated in soil under water-deficit conditions.The OE plants displayed lower rates of water loss from 1 h to 4 h after treatment compared with WT plants(Fig.5B),and showed a higher survival rate after 25 days without watering(Fig.5C).The OE plants under drought-stress conditions also showed lower MDA contents but higher proline and ABA contents than WT plants(Fig.5D-F).The expression of AtGolS2(encoding a stress-related galactinol synthase 2 protein)and AtDREB2C(encoding a dehydration-responsive element binding protein 2C)was induced in OE plants under both control and drought stress conditions,and OE plants showed higher transcription levels of AtP5CS1(encoding a delta 1-pyrroline-5-carboxylate synthase 1 protein),a proline biosynthesis gene,than WT plants under drought stress(Fig.5G-I).These results are similar to the previous finding that AtSAP5 affects the expression of abiotic stress-responsive genes in transgenic A.thaliana and cotton[38,39].Thus,there may be an association between overexpression of GmSAP5 and drought resistance in transgenic A.thaliana plants.

3.6.GmSAP5 confers resistance to drought stress in transgenic soybean hairy roots

Composite soybean plants consisting of non-transgenic leaves and transgenic hairy roots with overexpression or RNAi-mediated silencing of GmSAP5 were generated to further investigate how GmSAP5 contributes to the response to drought stress.There was no observable difference in growth phenotype between the composite plants harboring transgenic hairy roots and those harboring the empty vector(EV)control(Fig.6A,B).Compared with EV plants,the RNAi plants showed more severe leaf wilting after drought-stress treatment(Fig.6A).In contrast,OE plants showed increased resistance to drought stress,as reflected by a higher survival rate and larger RWC(Fig.6C,D).OE plants had higher contents of ABA and proline than EV and RNAi plants(Fig.6E,F).MDA contents in RNAi plants were markedly lower than those in both EV and OE plants(Fig.6G).These results suggested that GmSAP5 confers drought-stress resistance in soybean.

3.7.GmAREB3 activates the transcription of GmSAP5

To determine whether TFs are involved in the regulation of GmSAP5,Y1H assay was performed to screen for putative binding proteins of the GmSAP5 promoter.Using the GmSAP5 promoter as a bait in a yeast one-hybrid screen,GmAREB3/ABF3,a soybean homolog of A.thaliana DPBF3/AREB3,was isolated.Co-expression of the pB42AD-GmAREB3 construct with the pLacZi-ProGmSAP5construct induced the expression the LacZi reporter gene driven by the GmSAP5 promoter(Fig.7A).AREB/ABF TFs can bind to ABREs and have pivotal functions in the regulation of stress-responsive genes[40,41].To test whether GmAREB3 activates the expression of GmSAP5,we first analyzed the cis-active elements in the 2 kb region upstream of the 5′UTR of GmSAP5 and found two ABREs(Fig.S3),suggesting an interaction between the GmAREB3 protein and the GmSAP5 promoter.To determine whether GmAREB3 could activate the expression of GmSAP5,a transient luciferase transcriptional activity assay was performed in A.thaliana leaf mesophyll protoplasts.GmAREB3 greatly increased the expression of the luciferase reporter gene driven by the GmSAP5 promoter,and protoplasts expressing GmAREB3 showed markedly increased activation of the reporter gene in the presence of ABA(Fig.7B,C).These results suggest that GmSAP5 is regulated by GmAREB3.Transcripts of GmAREB3 were markedly increased by ABA or osmotic stress treatments(Fig.7D,E).Accordingly,K599 Agrobacteriummediated transformation of soybean hairy roots was performed to study the functions of GmAREB3.Overexpression of GmAREB3 increased drought-stress resistance in transgenic composite plants(Fig.S4).These results suggest that GmSAP5 increases droughtstress tolerance by mediating ABA signaling.

Fig.6.Phenotypic characterization of transgenic hairy root composite plants with overexpression(OE)or RNA interference(RNAi)-mediated silencing of GmSAP5 under drought stress conditions.(A)Soybean plants under drought-stress conditions.Water was withheld for 14 days and then plants were rehydrated for 3 days.(B)Relative transcript levels of GmSAP5 in RNAi,empty vector(EV),and OE plants determined by qRT-PCR.(C)Survival rates of seedlings were calculated from the results of three independent experiments.(D-G)Changes in relative water content(RWC)(D),ABA content(E),proline content(F),and MDA content(G)of seedlings.Asterisks indicate significant differences by two-tailed Student’s t-test(*,P＜0.05).

Fig.7.GmAREB3 activates the expression of GmSAP5.(A)The results of yeast-one-hybrid assays.(B,C)GmAREB3 activates GmSAP5 promoter-luciferase fusion constructs in transient transactivation assays.Asterisks indicate significant differences by two-tailed Student’s t-test(*,P＜0.05).(D,E)Expression patterns of the GmAREB3 gene under PEG 6000 and ABA treatments determined by qRT-PCR analysis.Values are means±SD of three technical replicates.

4.Discussion

SAPs that contain an N-terminal A20 domain and a C-terminal AN1 domain are rapidly induced by environmental stresses and have been identified[18,42,43]as key molecular factors responsible for protecting plants against abiotic stress.In previous studies[19-22,44],overexpression of SAP genes conferred stress tolerance in transgenic plants.In this study,we identified a GmSAP5 protein that was closely related to AtSAP5,TaSAP5,OSISAP1,and OsSAP11,proteins that function as positive regulators of abiotic stress response in plants as reported previously[18-22,38,39],suggesting that GmSAP5 acts in response to abiotic stresses.Transcription of GmSAP5 was induced by drought stresses(Fig.2),and GmSAP5 functions as a positive regulator of drought response by modulating the stomatal aperture and increasing primary root length,finally leading to a decrease in water loss rate and an increase in survival rate in transgenic A.thaliana plants under water-deficit conditions(Figs.3-5).Previous studies[45,46]have shown that a slow canopy wilting phenotype is associated with drought tolerance in soybean,and the slow-wilting phenotype trait is desirable in drought-tolerant crop breeding.Leaf RWC is seen[47]as a soybean physiological trait for drought selection.We observed larger RWC in OE composite soybean plants and more severe leaf wilt morphologies in RNAi soybean seedlings(Fig.6),indicating that overexpression of GmSAP5 can reduce water loss under water deficit conditions,and suggesting GmSAP5 as a candidate gene for soybean drought-tolerant genetic breeding.

Proline accumulation is a common physiological response to various environmental conditions,such as drought,salt,and oxidative stress.Proline is an inert compatible osmolyte that acts in maintaining turgor pressure and stabilizing cellular structures[48-50].There are close relationships between proline accumulation and osmotic stress tolerance in plants[51,52].ABA has been proposed[52,53]to be responsible for inducing proline accumulation through both the ABA-dependent and ABAindependent pathways in stressed plants.Proline accumulation then increases plant stress resistance by activating antioxidant enzymes,resulting in lower contents of ROS and MDA[51,54,55].In the present study,higher contents of ABA and proline and lower MDA contents were observed in OE plants under drought stress(Figs.5,6),and AtP5CS1,a proline biosynthesis gene,was induced in OE plants under drought-stress conditions,in accord with the changes in proline content(Fig.5E,I).These findings indicate that overexpression of GmSAP5 induces the accumulation of ABA and proline,which is accompanied by a reduction of MDA content,and finally confers stress tolerance under drought-stress conditions.

ABF/AREBs are TFs involved in ABA and drought response[35],and the ABRE is a cis element for ABA-responsive gene transcription under osmotic stress conditions[56].In our previous study,we identified a GmNF-YC14 TF that can interact with GmNF-YA16 and GmNF-YB2 to form a GmNF-YC14/B2/A16 complex,and confers drought stress tolerance by altering physiological processes.GmABF TFs(GmABF1-GmABF4)was induced in GmNF-YC14 overexpression lines under drought stress condition,and relative LUC activity assay indicated that GmABF3/AREB3 functions downstream of the GmNF-YC14/B2/A16 complex[41].Several studies[57-60]have shown that ABF/AREB TFs regulate the transcription of ABA-responsive genes in an ABRE-mediated manner and confer drought stress tolerance in plants.In the present study,yeast one-hybrid and transient transactivation assays showed that GmAREB3 binds to the promoter of GmSAP5,which contains two ABREs(Fig.7),and overexpression analyses showed that GmSAP5 and GmAREB3 both increased drought-stress tolerance in soybean plants(Figs.6,S4).These results suggest that GmSAP5 increases drought-stress tolerance via transcriptional regulation of ABA signaling-pathway genes.Overexpression of GmSAP5 in A.thaliana also affects the transcription of abiotic stress-responsive genes,which include GolS2 and DREB2C(Fig.5).Previous studies[18,38,39]have shown that SAPs can regulate the expression of stress-responsive genes in transgenic plants,suggesting that SAPs may act as transcriptional regulators under stress conditions.Further studies may shed light on the mechanisms by which GmSAP5 regulates osmotic stress responses.Overall,our results indicated that the GmNF-YC14/B2/A16 complex positively activates the transcription of GmAREB3 in soybean under drought stress[41],and the expression of GmSAP5 is activated by GmAREB3,finally causing stomatal closure.Proline acts as a protectant or osmolyte to reduce the membrane damage caused by osmotic stress.These changes finally increase drought-stress tolerance in plants(Fig.8).

Fig.8.Proposed model of GmSAP5-mediated drought response in soybean plants.ABA-PYL/PYR-PP2C-SnRK2 pathway controls ABRE-mediated transcription(ABF/AREB TFs)in drought-stress responses in plants[60-62].GmNF-YC14,GmNF-YB2 and GmNF-YA16 can form a complex to activate GmABF/AREB genes[41],and GmAREB3 binds to the promoter of GmSAP5 to activate its expression in response to drought stress in soybean.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Zehao Hou:Writing-original draft,Writing-review&editing.Xiangzhan Zhang:Writing-original draft.Yimiao Tang:Writing-review & editing.Taifei Yu:Writing-review & editing.LeiZheng:Writing-review&editing.Jun Chen:Methodology.Yongbin Zhou:Methodology.Yongwei Liu:Writing-review&editing.Ming Chen:Methodology.Zhao-Shi Xu:Funding acquisition,Project administration,Supervision.Youzhi Ma:Supervision.

Acknowledgments

This research was supported by the National Natural Science Foundation of China(31871624),the Agricultural Science and Technology Innovation Program(CAAS-ZDRW202109 and CAASZDRW202002),the Central Public-interest Scientific Institution Basal Research Fund(S2022ZD02)and the National Transgenic Key Project of the Chinese Ministry of Agriculture(2020ZX08009-15B).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.03.013.