A GmSIN1/GmNCED3s/GmRbohBs Feed-Forward Loop Actsas a Signal Amplifier That Regulates Root Growth in SoybeanExposed to Salt Stress[OPEN]

Shuo Li,a,1 Nan Wang,a,1 Dandan Ji,a,1,2 Wenxiao Zhang,a,1 Ying Wang,a Yanchong Yu,a,3 Shizhen Zhao,a

Menghua Lyu,a Juanjuan You,a Yangyang Zhang,a Luli Wang,a Xiaofang Wang,a Zhenhua Liu,a Jianhua Tong,b

Langtao Xiao,b Ming-yi Bai,a and Fengning Xianga,4

a The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences,Shandong University, Qingdao 266237, People’s Republic ChinabHunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Provincial Key Laboratory for CropGermplasm Innovation and Utilization, Hunan Agricultural University, Changsha 410128, People’s Republic of China

ORCID IDs: 0000-0002-7285-8407 (S.L.); 0000-0001-5101-6238 (N.W.); 0000-0002-8209-4578 (D.J.); 0000-0002-8356-6370 (W.Z.);0000-0002-0269-2894 (Y.W.); 0000-0002-5389-1922 (Y.Y.); 0000-0002-8187-1857 (S.Z.); 0000-0001-9952-7512 (M.L.); 0000-0002-6211-2812 (J.Y.); 0000-0002-7277-9610 (Y.Z.); 0000-0002-2350-7889 (L.W.); 0000-0002-9790-3032 (X.W.); 0000-0002-9752-7356(Z.L.); 0000-0001-9785-9652 (J.T.); 0000-0003-1786-1950 (L.X.); 0000-0001-5992-7511 (M.-y.B.); 0000-0002-8334-3273 (F.X.)

Abscisic acid (ABA) and reactive oxygen species (ROS) act as key signaling molecules in the plant response to salt stress;however, how these signals are transduced and amplified remains unclear. Here, a soybean (Glycine max) salinity-inducedNAM/ATAF1/2/CUC2 (NAC) transcription factor encoded by SALT INDUCED NAC1 (GmSIN1) was shown to be a keycomponent of this process. Overexpression of GmSIN1 in soybean promoted root growth and salt tolerance and increasedyield under salt stress; RNA interference–mediated knockdown of GmSIN1 had the opposite effect. The rapid induction ofGmSIN1 in response to salinity required ABA and ROS, and the effect of GmSIN1 on root elongation and salt tolerance wasachieved by boosting cellular ABA and ROS contents. GmSIN1 upregulated 9-cis-epoxycarotenoid dioxygenase coding genesin soybean (GmNCED3s, associated with ABA synthesis) and Respiratory burst oxidase homolog B genes in soybean(GmRbohBs, associated with ROS generation) by binding to their promoters at a site that has not been described to date.Together, GmSIN1, GmNCED3s, and GmRbohBs constitute a positive feed-forward system that enables the rapidaccumulation of ABA and ROS, effectively amplifying the initial salt stress signal. These findings suggest that thecombined modulation of ABA and ROS contents enhances soybean salt tolerance.

INTRODUCTION

High salt concentrations in soils strongly reduce crop perfor-mance (Munns andTester, 2008) , and salt-affected soils currentlyaccount for 10%of theworld’s total arable land area (Shahid et al.,2018).Soybean (Glycinemax) isamajoragricultural crop foroil andprotein resources, and its production is reported to decrease by40% with increasing salt stress (Papiernik et al., 2005) . To en-gineersalt-tolerant soybeanvarieties, it iscrucial to identify thekeycomponents of the plant salt-tolerance network. Many soybeangenes (Wanget al., 2016), including transcription factor (TF) genes(Chen et al., 2007; Wei et al., 2009; Zhang et al., 2009; Hao et al.,

2011; Zhai et al., 2013; Wang et al., 2015a), have been found toconfer salt stress tolerance when heterologously expressed intransgenicmodel plants. In soybean, a cation/H1 exchanger genecontributes to natural variation in salt tolerance (Guan et al., 2014;Qi et al., 2014). However, few of these genes have been dem-onstrated to affect soybean yield in saline field conditions andlittle is known about how soybeans respond and adapt to highsalinity (Shi et al., 2018; Zhang et al., 2019). This lack ofknowledge has greatly inhibited attempts to enhance salt tol-erance in crops.Understanding how plants generate and transduce salt stress

signals could provide novel approaches for enhancing salt toler-ance. In theplant response tohighsalinity, the initial signal activatesthe production of compounds that trigger activity in a number ofmetabolicanddevelopmental pathways.Oneof themost importantcompounds is abscisic acid (ABA; Finkelstein and Gibson, 2002).Under salt or drought stress conditions, the endogenousplant ABAlevel can rise by ;40-fold (Verslues et al., 2006).Activation of ABA biosynthetic enzymes has a key role in salt

and drought stress responses. During ABA biosynthesis, thecleavage of 9-cis-epoxycarotenoids to xanthoxin is catalyzed by9-cis-epoxycarotenoid dioxygenases (NCEDs); this is believed tobe the key regulatory step of ABA biosynthesis (Lefebvre et al.,2006). Among the five NCED genes in Arabidopsis (Arabidopsis

1 These authors contributed equally to this work.2 Current address: College of Environmental Science and Engineering,Qilu University of Technology (Shandong Academy of Science), Jinan,Shandong, China 250353.3 Current address: College of Life Sciences, Qingdao AgriculturalUniversity, Qingdao 266109, China.4 Address correspondence to xfn0990@sdu.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Fengning Xiang(xfn0990@sdu.edu.cn).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.18.00662

The Plant Cell, Vol. 31: 2107–2130, September 2019, www.plantcell.org ã 2019 ASPB.

thaliana),NCED3playsakey role inABAbiosynthesis duringwaterdeficit and salt stress (Iuchi et al., 2001; Tan et al., 2003; Nambaraand Marion-Poll, 2005; Barrero et al., 2006). The Arabidopsis TFNGATHA1 induces NCED3 in response to drought stress (Satoet al., 2018); however, the factors that induce NCED genes inresponse to salt stress remain largely unknown.

Reactive oxygen species (ROS) are also implicated in the saltstress response (Yang and Guo, 2018). ROS participate ina number of signaling pathways and other processes, but ROSover-accumulation is cytotoxic (Mittler, 2017). The ROS super-oxide is generated by respiratory burst oxidase homologs(RBOHs), a family of proteins that are well conserved throughoutthe plant kingdom. The rapid production of ROS generated byNADPH oxidases (NOXs) occurs in response to a number of ex-ternal stimuli (Marino et al., 2012). Analysis of the ArabidopsisatrbohD mutant has suggested that RbohD is required togenerate ROS in plants exposed to salt stress (Xie et al., 2011).ABA influences the production of ROS through its effect onRBOH expression or RBOH activity (Kwak et al., 2003; Lin et al.,2009). Therefore, ABA and salinity signaling are thought tooverlap by both affecting RBOH-derived ROS production (Xieet al., 2011).

The relationship between ABAandROS in regulating salt stressresponses and plant growth is far from fully understood (Mittlerand Blumwald, 2015; Qi et al., 2018). ABA clearly stimulates theproduction of ROS in Arabidopsis guard cells (Zhang et al., 2001;Wang and Song, 2008; Jannat et al., 2011) and maize (Zea mays)leaves (Jiang and Zhang, 2002). However, the situation in rootsremains unclear as some studies have found that ABA promotesROS production in roots (Kwak et al., 2003; He et al., 2012; Jiaoet al., 2013; Yang et al., 2014), but others show that ABA sup-presses ROS production in roots (Almagro et al., 2009; Zhanget al., 2014).

Many attempts have been made to enhance plant stress tol-erance by modulating ABA/ROS levels or signaling. Indeed, en-hanced ABA or ROS levels or signaling promotes abiotic stresstolerance in transgenic plants (Min et al., 2015; Yang et al., 2015b;Perea-Resaetal., 2016;Qinetal., 2016;Fanetal., 2019).However,in other cases, lower ABA or ROS levels elevate abiotic stresstolerance (Ruggiero et al., 2004; Wu et al., 2014; Shu et al., 2015;Yang et al., 2015a). It is also notable that the enhancementof abiotic stress tolerance in transgenic plants with improvedABA signaling is often coupled with a negative effect on growth(Fujita et al., 2005; Furihata et al., 2006; Wang et al., 2015b).Therefore, the complex interaction between ABA and ROS inabiotic stress responses needs to be clarified to enable im-provements of plant stress tolerance by modulating ABA andROS levels/signaling.

The NAM/ATAF1/2/CUC2 (NAC) genes encode plant-specificTFs (Riechmann et al., 2000) and comprise one of the largest TFfamilies in plants, with 106 NAC genes in Arabidopsis and 226 insoybean (Olsen et al., 2005b; Le et al., 2011) . NAC TFs playimportant roles in various biological processes including the saltstress response; for example, overexpression of specific NACTF genes can improve salt tolerance in plants (Jeong et al., 2010;Hao et al., 2011; Han et al., 2015; Huang et al., 2015). SomeNAC-specific binding motifs have been shown to mediate thedirect transcriptional regulation of their target genes (listed

in Supplemental Table 1). This suggests that the diversity of NACbinding motifs is related to the functional specificity of NAC TFs.However, our knowledge of the binding motifs and direct targetgenes of the NAC TFs in salt stress responses remains limited,particularly in legume crops. Identification of NAC binding sitesand NAC target genes will uncover the transcriptional regulatorynetworks of specific NAC TFs that function in salt stressresponses.Approaches to identify genes related to salt tolerance include

examining cultivars that showstrongsalt toleranceand identifyinggenes inducedby salt stress. To this end, earlierwork selected thesoybeancvShengdouNo. 9 as themost salt-tolerant accession ina panel of cultivars (Ji et al., 2011). A microarray assay comparingsoybean cv Williams 82 with and without salt stress identifieda number of salt stress–induced genes; someof these geneswereisolated and tested to see whether their overexpression couldconfer salt tolerance (Song et al., 2012). One highly salt-inducedgene (up to 120-fold induction) was cloned from Shengdou No. 9(Ji et al., 2011) and named SALT INDUCED1 (GmSIN1). GmSIN1encodes a predicted NAC-domain TF and overexpression ofGmSIN1 led to a marked improvement in field-based salinitytolerance. Here, we investigated the effect of overexpressing andknocking downGmSIN1 on root growth and salt tolerance. Basedon the results, we propose a feed-forward pathway in whichGmSIN1, GmNCED3s, and GmRbohBs collaborate to rapidlyamplify the initial salt stress signal.

RESULTS

GmSIN1 Promotes Root Growth and Salt Tolerance

To confirm the function of GmSIN1 in soybean salt tolerance, wegenerated transgenic soybean lines that overexpressed or si-lenced GmSIN1. We constructed GmSIN1 overexpression (OE;35Spro:GmSIN1) and RNA interference (RNAi; 35Spro:GmSIN1RNAi) transgenic soybean lines in the salt-tolerant cv Wei6823(Supplemental Figures 1 and 2), and the T4 homozygousOE lines(Supplemental Figure 3) and T2RNAi lineswere examined for theirsalt tolerance.To test the effect of overexpression and silencing of GmSIN1,

we first examined the effect on root growth. In bag-grownseedlings grown under normal lab growth conditions, the OElines produced longer primary roots compared with Wei6823(Figures 1A and 1B; Supplemental Figure 4), and RNAi linesproduced shorter primary roots (Figures 1C and 1D). Under NaClstress (150mM, treatmentof3-d-oldseedlings for4d), theprimaryroots were longer in OE lines and shorter in RNAi lines comparedwith the wild type. Therefore, in terms of root growth, theOE linesshowedmoresalt tolerancecomparedwithwild typeand theRNAilines showed less salt tolerance (Figures 1A to 1D; SupplementalData Set 1).We next examined other cellular processes affected by salt

stress, specifically photosynthetic parameters and antioxidantdefenses. For photosynthesis, we measured the net photosyn-thesis rate, stomatal conductance, intercellular CO2 concentra-tion, and photosystem II photochemical potential. These weremeasured in the first trifoliolate compound leaves of 2-week-old

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Figure 1. GmSIN1 Promoted Root Growth and Salt Tolerance.

(A) to (D)Root lengthof7-d-oldsoybeanseedlingschallengedwitheither0mM(mock)or150mMNaCl. Imageswere taken4dafter thestress treatmentwasinitiated (A) and (C). Each column represents themean6 SE length of 20 to 30 roots (B) and (D). The roots were collected from seedlings grown in 10 plasticgrowth bags with the same treatment. Each bag contained three seedlings each from the wild-type (WT) and three transgenic lines. The data shown arerepresentative of several independent experiments. G, genotype; T, treatment; G 3 T, genotype 3 treatment. Bar 5 5 cm.(E) and (F)Emergence rate (see [F];n56experimentswith 40 seeds fromeach genotypeper experiment) and plant height (see [E];n560, 10plants in eachof 6 replicate plots) of the wild type (WT) and three independentGmSIN1 overexpressors (OE-1,OE-2, andOE-3) grown in the field under the same climateenvironment but with different salinity. For emergence rate, data were collected from the replicate plots. In each plot, 40 seeds were planted for eachgenotype. For height assays, 10 seedlings of each line from each plot were randomly selected. The salinity of the control field and saline field were 0.15 and0.35 g of total soluble salts per 100 g of dry soil, respectively. Each column represents the mean emergence rate or plant height 6 SE. G, genotype; T,treatment; G 3 T, genotype 3 treatment.

GmSIN1 in Root Growth and Salt Tolerance 2109

Wei6823 and OE plants at 10 d after a 2-d treatment with 50, 75,100, 125, and150mMNaCl. The photosynthesis ofOEplantswasreduced to a lesser extent than that of thewild type (SupplementalFigures 5A to 5D). Next, we compared the concentrations of Proand malonaldehyde and the activity of the antioxidant enzymessuperoxide dismutase, peroxidase, and catalase under NaClstress between the OE lines and Wei6823 (Supplemental Figures5E to 5I). After NaCl treatment, the GmSIN1 OE lines showedhigher accumulation of Pro, lower concentrations of malonalde-hyde, and higher activities of ROS-scavenging enzymes com-pared with Wei6823.

We next examined salt tolerance in field conditions. For this, weselected three single-copy GmSIN1 OE homozygous transgeniclines.Acomparisonof thefieldperformanceof theOE linesand thewild typeunder control (0.15gof total soluble salts per 100gof drysoil) and saline (0.35 g of total soluble salts per 100 g of dry soil)conditions (Supplemental Figure 6A) in the same environmentshowed that for all threeOE lines, seedlings emergedmore readilythan did the wild type (Figure 1E; Supplemental Data Set 1). Theheight of mature wild-type plants was strongly reduced in salinesoil, but theeffecton theOE lineswasmuch lesssevere (Figure1F;Supplemental Data Set 1). Further yield trials were conducted inthree different geographical locations using fields with 0.05, 0.30,and0.35gof total soluble salts per 100gof dry soil. In all fields, theOE plants were taller than thewild type, producedmore seeds perplant, and had higher yields (Figure 1G; Supplemental Figure 6B;Supplemental Data Set 1). Over four seasons, the performance oftheOE lines undermedium-salinity conditions (0.2 to 0.3 g of totalsoluble salts per 100 g of dry soil) exceeded that of thewild type interms of plant height, pod number per plant, and the number ofseeds per plant (Table 1). Therefore, GmSIN1 overexpressionenhanced plant growth and productivity not just under salineconditions but also (albeit to a lesser extent) under nonsalineconditions.

Taken together, these findings suggested that GmSIN1 influ-ences root growth and salt tolerance in soybean.

Characterization of GmSIN1

Asapreliminary characterizationofGmSIN function,weexaminedits conserved domains and subcellular localization. In silicotranslation of the GmSIN1 coding sequence predicted a 342-residue polypeptide including a conserved NAC domain (aminoacids 14 to 139) in the N terminus and a variable C terminus(Supplemental Figure 7A). GmSIN1 shares amean identity of 45%with its Arabidopsis homologs (Supplemental Figure 7A). Aphylogenetic tree based on polypeptide sequences showed thatGmSIN1 is closely related to Glyma.13G279900 and Glyma.

12G221500 from soybean andANAC019, ANAC055, andANAC072from Arabidopsis (Supplemental Figure 7B; Supplemental DataSet 2). GmSIN1 shares a mean identity of 45% with its Arabi-dopsis homologs (Supplemental Figure 7A).The subcellular distribution of GmSIN1 was investigated

by imaging a GmSIN1-green fluorescent protein (GFP) fusiontransformed into Arabidopsis protoplasts (Supplemental Figure 8).In these protoplasts, signal was detectable in the nucleus(Figure 2A).To further characterize GmSIN1 function, we examined its

transcriptional activation activity by fusing its coding sequencewith the GAL4 DNA binding domain of the yeast expressionvector pGBKT7 and tested whether this fusion could activatereporter genes (conferringHis auxotrophy andb-galactosidaseactivity) downstream of the GAL4 binding site. The yeasttransformants with pGmSIN1-GBKT7 or the empty vector grewfreely onmedium lacking Leu (Figure 2B). However, onmediumlacking His, cells having the empty vector pGBKT7 did notgrow, while those carrying GmSIN1 grew well (Figure 2B). Thetransactivation activity assay (X-b-Gal assay) gave a consistentresult (Figure 2B). Therefore, GmSIN1 represents a soybeangene that encodes a nucleus-localized NAC transcriptionactivator.

Figure 1. (continued).

(G)Representative results of 10 seedlings each forGmSIN1OE-1,OE-2,OE-3, and thewild type (WT) grown in different fieldswith different salinity, and theaverage number of seeds produced per seedling. Numbers indicate the average number of seeds6 SE (n5 30). Five seedlings of each line from each plotwere randomlyselected.Valuesat the top indicate thesoil salinity (total solublesaltsamountper100gofdryweightofsoil). Two-wayANOVAwasconductedin (B), (D), and (F) and genotype and NaCl treatment as the two factors. The P-values are shown. Significant differences between samples labeled withdifferent Roman (a, b) or Greek letters (a, b, g, d) were determined by one-way ANOVA, P < 0.05. ND, no significant difference (the details of the statisticalresults are in Supplemental Data Set 1). G, genotype; T, treatment; G 3 T, genotype 3 treatment.

Table 1. Traits of GmSIN1 OE Transgenic Seedlings in Medium-SalineField

Year Line Height Effective Pod No. Seed No.

2018 GmSIN1 OE-1 68 6 3*** 66 6 3** 121 6 6**GmSIN1 OE-2 69 6 3*** 69 6 4** 114 6 6*GmSIN1 OE-3 69 6 2*** 64 6 3* 108 6 5WT 54 6 2 56 6 2 96 6 6GmSIN1 OE-1 61 6 6** 157 6 42*** 276 6 33***

2016 GmSIN1 OE-2 63 6 10** 90 6 17*** 147 6 26GmSIN1 OE-3 61 6 8** 104 6 19*** 207 6 35***WT 51 6 8 62 6 11 124 6 26

2015 GmSIN1 OE-1 55 6 6*** 145 6 37* 286 6 88**GmSIN1 OE-2 53 6 8** 136 6 36* 261 6 58**WT 43 6 7 105 6 44 191 6 72

2014 GmSIN1 OE-1 63 6 4* 40 6 7* 74 6 36*GmSIN1 OE-2 58 6 5* 40 6 9 85 6 37*GmSIN1 OE-3 57 6 4 45 6 9* 80 6 39*WT 43 6 4 32 6 5 66 6 34

The significance of differences between GmSIN1 OE transgenic and thewild-type (WT) seedlings was determined with the Student’s t test (*P <0.05; **P < 0.01; ***P < 0.001, n 5 15 to 45 plants). All data are given asmean 6 SE. The salinity of the field is ;0.2 to 0.3 g of soluble salt per100 g of dry soil.

2110 The Plant Cell

We further investigated the expression pattern of GmSIN1 byquantifying the relativeabundanceof themRNA indifferentorgansof Shengdou No. 9 plants. The GmSIN1 expression was muchhigher in roots, but moderate in stems, leaves, and flowers. Thetranscripts were not detectable in seeds (Figure 2C). Using RNAin situ hybridization, we detected the expression of GmSIN1predominantly in endodermal cells and cells associatedwith phloemand xylem in the root (Figure 2D). Similar to other stress-responsiveNACTFgenes (Leetal., 2011), theexpressionofGmSIN1wasrapidlyinduced by high salinity, osmotic stress (induced by polyethylene

glycol treatment), cold, oxidative stress, and ABA treatment in thesalt-tolerant cv Shengdou No. 9 (Figure 2E).

Transcriptomic Analysis of 35Spro:GmSIN1Transgenic Soybeans

To reveal the effect of the TF GmSIN1 on the soybean tran-scriptome in response to salt stress, we conducted RNA se-quencing (RNA-seq) on theOE andwild-type plants under controlconditions (mock treated) and NaCl treatment. We first tested the

Figure 2. Characterization of GmSIN1.

(A) GmSIN1-GFP fusion proteins localized to the nucleus in transiently transformed Arabidopsis protoplasts. (Left) Images of a protoplast harboringp35Spro:GmSIN1-GFP and pMDC32-1ABES1n-mCherry (nuclearmarker). (Right) Images of a protoplast harboring p35Spro:GFP. The top row shows theGFP signal (green), the middle row shows the nuclear marker (left) and chloroplast autofluorescence (right; magenta), and the bottom row shows themerged images. The images show representative results frommore than 30 transformed protoplasts from three independent experiments.More imagescan be found in Supplemental Figure 8B. Bar 5 20 mm.(B) Ability of yeast transformants to grow onmedium lacking His and Leu but containing 10 mM 3-aminotriazole, and the formation of color in the X-b-Galassay indicates transcriptional activation. The images show representative results frommore than five independent yeast transformants. BD, Yeast colonyexpressing GAL4 DNA binding domain; BD-GmSIN1, Yeast colony expressing BD-GmSIN1 fusion protein.(C) Expression of GmSIN1 analyzed using RT-qPCR in root, stem, leaf, flower, and seed tissue in cv Shengdou No. 9.(D) Localization ofGmSIN1mRNA using in situ hybridization. (Left) Transverse section of a root 1mm from the root tip of 6-d-old cv ShengdouNo. 9 plantsprobed with a GmSIN1 antisense probe. (Right) Profile obtained using a sense strand probe. Bar 5 100 mm.(E) Expression of GmSIN1 analyzed using RT-qPCR in response to NaCl (150 mM), moisture stress (20% [w/v] polyethylene glycol [PEG] 6000), lowtemperature (4°C), 100 mMABA, or 1mMH2O2 treatment in cv Shengdou No.9. The roots of 2-week-old seedlings were collected for RNA extraction. Errorbars represent SE (n 5 3 biological repeats). The details of the sampling methods are shown in “Methods.”

GmSIN1 in Root Growth and Salt Tolerance 2111

Figure 3. Transcriptomic Analysis of GmSIN1 OE-1 Transgenic Soybean.

(A)and (B)Numbersofgenesshowingdifferential expressionbetweenGmSIN1OE-1 transgenic soybeansandWei6823 (A)orbetweennon-NaCl–stressedand NaCl-stressed seedlings (B). WT, wild type.

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expression pattern of GmSIN1 in the wild type and OE-1 in re-sponse to 150 mM NaCl treatment. In both the wild-type and OEplants, GmSIN1 was induced rapidly in response to NaCl stressand achieved peak expression at 6 h. After that, GmSIN1 ex-pression declined quickly and reached its original level at 48 h.During the treatment, the expression level of GmSIN1 was higherin the OE plants than in the wild-type plants (SupplementalFigure 9A). This suggests that GmSIN1 mainly functions in theearly response to salt stress.

Based on the expression of GmSIN1, we used a 6-h NaCltreatment in the RNA-seq experiment and performed two bi-ological replicates (Supplemental Table 2). Following the 6-hNaCl treatment, 2370 genes were differentially expressed (foldchange > 2 and P > 0.7) in the wild type relative tomock treatment(Figure 3A; Supplemental Data Set 3). However, NaCl treatmentcaused a less dramatic transcriptomic change in GmSIN1 OEplants relative to the wild-type plants, with only 363 upregulatedgenes and 316 downregulated genes (Figure 3A; SupplementalData Set 3). This suggests that GmSIN1 OE plants are less sen-sitive to NaCl treatment than the wild-type plants in terms oftranscriptomic changes.

The largest number of differentially expressed genes (DEGs),4357, was found between mock-treated GmSIN1 OE plants andthe wild-type plants. This included 609 DEGs in common withDEGs from the comparison of the NaCl-treated and control wild-type plants (Figure 3B; Supplemental Data Set 3). To betterunderstand the relationship of DEGs caused by GmSIN1 over-expression (DEGs inGmSIN1OEmock versus thewild-typemock)and by NaCl treatment (DEGs in the wild-type NaCl treatmentversus the wild-type mock), we analyzed the expression pattern ofthe 609DEGsusing hierarchical clustering andcorrelation analysis.This showed that for most DEGs, the expression pattern is exactlythe same (Figure 3C) and the correlation is as high as 0.96(Figure 3D). These results suggest that some of the transcriptionalchanges caused by salt stress are mediated by GmSIN1.

Biological pathways of secondary metabolites, especially forthe biosynthesis of flavonoid, flavone, flavonol, and isoflavonoidcompounds, were greatly enriched among the upregulated DEGsin mock-treated GmSIN1 OE plants versus the wild-type plants.Moreover, the

gene ontology (GO) terms responsive to stress, antioxidantactivity, hydrogen peroxide metabolic, and ABA metabolic wereespecially enrichedamong theupregulatedgenes (Figures 3Eand3F). This was consistent with the conclusion that GmSIN1 is

involved in the salt stress response. Moreover, it suggesteda function for GmSIN1 in ABA and ROS homeostasis.To further explore the effect of GmSIN1 on the transcription of

ABA- andROS-relatedgenes,we identifiedall the genes thatwerehomologs of Arabidopsis genes annotated as functioning in ABAbiosynthesis, ABA signaling, ROS generation, and ROS scav-enging. This identified 5 genes in ABA biosynthesis and signaling,4 genes in ROS generation, and 32 genes in ROS scavenging thatwere significantly upregulated in GmSIN1 OE-1 plants in com-parison with the wild type (fold change > 2 and P > 0.7) undernormal conditions (Figure 3G). We then hypothesized thatchanges in the expression of these genesmay affect the ABA andROS levels and contribute to salt tolerance of GmSIN1 OE soy-bean. To test this, three ABA- or ROS-responsive genes wereselected and their expression was measured using RT-qPCR inGmSIN1 OE-1 and RNAi plants (Supplemental Figure 9B). Theselected genes were all upregulated in OE plants and down-regulated in RNAi plants (Supplemental Figure 9C). Additionally,they all showed earlier or higher induction inOE plants than in thewild-type plants in response to NaCl treatment (SupplementalFigure 9D). These data further suggest that GmSIN1 affects ABAand ROS pathways at early stages of NaCl treatment.

GmSIN1 Induces the Production of ABA and ROS inResponse to Salt Stress

NCED3 and NOX have been suggested to be responsible for theincreases in ABA and ROS that occur in response to salt stress inArabidopsis (Barrero et al., 2006; Kurusu et al., 2015). NCED3 andNOX genes were upregulated in the GmSIN1 overexpressionlines, as shown by the RNA-seq data (Figure 3G). Therefore,we tested the effect of GmSIN1 overexpression and suppressionon GmNCED3-1 (Glyma.15G250100.1), GmNCED3-2 (Glyma.08G176300.1),GmRbohB-1 (Glyma.10G152200.1), andGmRbohB-2 (Glyma.20G236200.1) transcripts in the presence of salt stressby RT-qPCR of OE and RNAi plants. Under control (nonsaline)conditions, the gene expression in the OE seedlings was higherthan in the wild-type seedlings that, in turn, was higher than inRNAi seedlings (Figure 4A). The abundance of each transcriptincreased in the roots of thewild-type andOE seedlings exposedto 150 mM NaCl for 6 h, but not in the RNAi plants (Figure 4A).To demonstrate the functionality of GmNCED3-1 and

GmRbohB-1 in soybean, the two genes were transiently over-expressed in soybean leaves. Indeed,GmNCED3-1 overexpression

Figure 3. (continued).

(C) Hierarchical cluster analysis of the overlapping genes from (B) differentially expressed in NaCl-treated Wei6823 versus mock-treated Wei6823 andGmSIN1 OE-1 versus Wei6823. The numerical values in the yellow-to-blue gradient bar represent log2-fold change relative to the control sample. WT,wild type.(D)Correlation of the overlapping genes from (B)differentially expressed inNaCl-treatedWei6823 versusmock-treatedWei6823 andGmSIN1OE-1 versusWei6823. Cor, correlation; WT, wild type.(E)Pathways thatwerestatistically enriched inDEGs inGmSIN1OE-1versusWei6823RNA-seqdata. Thenumbersnear thecolumns indicate thenumberofDEGs with corresponding annotation and the P-value, respectively.(F) GO terms that were statistically enriched in differentially expressed genes in GmSIN1 OE-1 versus Wei6823 RNA-seq assay. The numbers near thecolumns indicate the number of DEGs with corresponding annotation and the q value, respectively.(G) Heatmap of differential expression of ABA biosynthesis, ABA signaling, ROS generation, and ROS scavenging-related genes inGmSIN1 OE-1 versusWei6823. The numerical values for the yellow-to-blue gradient bar represent log2-fold change relative to the control sample. WT, wild type.

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Figure 4. GmSIN1 Modulated ABA and ROS Contents by Inducing Genes Involved in ABA/ROS Synthesis in Response to Salt Stress.

(A) Transcript levels of GmNCED3-1, GmNCED3-2, GmRbohB-1, and GmRbohB-2 in the roots of 6-d-old seedlings of GmSIN1 OE (OE-1), a GmSIN1knockdown (RNAi ), and the wild type (WT) exposed to either 0 mM (mock) or 150 mM NaCl for 6 h. Data obtained by RT-qPCR.(B) ABA content of the root and leaf of the 5-d-old seedlings shown in (A). WT, wild type.

2114 The Plant Cell

had a positive effect on ABA accumulation and GmRbohB-1overexpression similarly increased ROS generation (SupplementalFigure 10). The effect of altering the abundance of GmNCED3-1,GmNCED3-2, GmRbohB-1, and GmRbohB-2 transcripts on theABA and ROS contents was explored by analyzing root and leafsamples fromGmSIN1OE andRNAiplants grown in the presenceorabsenceofsalt stress.The levelofABA inGmSIN1OE rootswassignificantly higher (fivefold and threefold higher for mock andNaCl treatment, respectively) than in the wild-type roots, and inGmSIN1RNAi roots, theopposite trendwasobserved (Figure 4B).In leaves, the change of ABA level was similar to that in roots(Figure 4B). Comparing the ABA increase in response to 6-h salttreatment in the wild-type and GmSIN1 RNAi plants showed thatthe ABA increasewas partially suppressed inGmSIN1RNAi rootsand completely suppressed in leaves. This suggested that theinduction of ABA under short-term salinity in soybeanwas at leastpartially mediated by GmSIN1.

Using nitroblue tetrazolium (NBT) to quantify the production ofthe ROS superoxide, we observed that the overexpression ofGmSIN1 promoted the accumulation of superoxide in the root.Thestaining intensitywasmuchweaker in thewild-type roots, andeven weaker in the RNAi roots compared with the OE roots(Figure4C).When theplantswereexposed to150mMNaCl for2h,superoxideproductionwasconcentratedat the root tipof thewild-type plants but at a lower level than in theOE roots and at a higherlevel than in the RNAi roots (Figure 4C). Greater NOX activity wasseen in theOE tissues than in thoseof thewild type, and lessNOXactivity was seen in RNAi tissues (Figure 4D). Finally, the cellularH2O2 content, as measured by 3,3-diaminobenzidine (DAB)staining, was higher in OE than in the wild type and lower inRNAi tissue (Figure 4E). The overexpression ofGmSIN1 promotedH2O2 accumulation under saline and nonsaline conditions in boththe root and the leaf (Figure 4F). The induction of superoxide,H2O2, andNOXactivity in response toNaCl treatment inGmSIN1RNAi lines was partly suppressed in comparison with the wildtype. Overall, this suggests that GmSIN1 contributes to salinity-induced ROS accumulation at early stages in response to saltstress.

GmSIN1 Improves Root Growth and Salt Tolerance byPromoting ABA Accumulation and ROS Generation

To investigate whether altered root growth and salt tolerance intransgenic soybean were due to changes in ABA levels and/orROS accumulation, we used diphenyliodonium chloride (DPI), an

inhibitor of NADPH oxidases, which contribute to the NaCl-induced ROS accumulation (Mazel et al., 2004), and nordihy-droguaiaretic acid (NDGA), an inhibitor of lipoxygenase, whichcatalyzes dioxygenation of polyunsaturated fatty acids and isreported to inhibit carotenoid cleavage dioxygenases includingNCED (Creelman et al., 1992) in root elongation assays inGmSIN1OE and the wild-type plants. Compared with the wild type,GmSIN1 OE lines produced longer primary roots under optimalgrowth conditions and significantly more relative root elongationafter a 4-d treatment with 150 mM NaCl (Figure 1A). Surprisingly,when the NaCl was coupled with DPI or NDGA treatment, thedifference of root length and root elongation between OE plantsand the wild-type plants was abolished (Figures 5A to 5C). Thisdemonstrates that the salt tolerance conferred by GmSIN1requires both ROS and ABA.Furthermore, DPI or NDGA treatment completely rescued the

long-root phenotype in GmSIN1 OE plants under optimal con-ditions (Figures 5D and 5E). Fluridone (FLU), an inhibitor of ca-rotenoid synthesis that has also been linked to depressed levelsof endogenous ABA, has a similar effect to NDGA (Figure 5F;Supplemental Figure 11). Therefore, the salt tolerance in rootelongation and the promotion of root length inGmSIN1 OE plantsrequired theaccumulation of bothABAviaNCEDactivity andROSvia NOX activity. These data also demonstrate that the genesencoding NCED and NOX function genetically downstream ofGmSIN1 in this process.GmSIN1 is transcriptionally induced by NaCl, oxidative stress,

and ABA treatment. To determine whether its induction is medi-ated by ABA or ROS, we used FLU or DPI to inhibit ABA orROS production in NaCl-treated seedlings, respectively. Indeed,GmSIN1 induction by high salinity was almost completely sup-pressed by FLU and DPI (Figure 5F). Additionally, the induction ofGmSIN1 by ABA was suppressed by DPI treatment and the in-duction of GmSIN1 by H2O2 was suppressed by FLU treatment(Figure 5F).In addition, weconstructedRNAi vectors that targetGmNCED3

andGmRbohB. These two constructs and the control vector weretransiently expressed in Shengdou No. 9 leaves, and GmSIN1expression was measured using RT-qPCR with or withoutNaCl treatment. The induction of GmSIN1 in response to NaCltreatment was significantly reduced when the expression ofGmNCED3 and GmRbohB was suppressed (Figure 5G). Theevidence strongly suggests that the high induction of GmSIN1in response to NaCl treatment is mediated by ABA and ROS,both of which are required for this response.

Figure 4. (continued).

(C)NBT staining reveals the superoxide content of primary and lateral roots of 6-d-old and of leaves of 17-d-oldGmSIN1OE (OE-1), aGmSIN1 knockdown(RNAi ), and the wild-type (WT) seedlings exposed to either 0 mM (mock) or 150 mM NaCl for 2 h. The staining intensity reflects the concentration ofsuperoxide.(D) NOX activity in the OE-1, RNAi, and the wild-type (WT) plants shown in (C). FW, fresh weight.(E)DABstaining reveals theH2O2 content of the leaf of 17-d-oldGmSIN1OE-1,GmSIN1RNAi, and thewild-type (WT) plants exposed to either 0mM (mock)or 150 mM NaCl for 2 h. The staining intensity reflects the concentration of H2O2.(F) The H2O2 content of the root of 6-d-old and the leaf of 17-d-old OE-1, RNAi, and the wild-type (WT) seedlings. Histogram data are given in the formmean6 SE (n5 3 for RT-qPCR data and n5 9 to 12 for the other assays). Significant differences between samples labeled with different Roman (a, b, c) orGreek letters (a, b, g) were determined by one-way ANOVA and Tukey’s test, P < 0.05. The details of the sampling procedures are presented in the“Methods.” FW, fresh weight; ND, no significant difference.

GmSIN1 in Root Growth and Salt Tolerance 2115

Figure 5. ABA and ROS Are Required for GmSIN1 to Enhance Salt Tolerance and Root Growth.

(A) Phenotype of 7-d-oldGmSIN1 OE transgenic plants compared with Wei6823 under control, 150 mMNaCl, 150 mMNaCl with 100 mMDPI, or 150 mMNaCl with 50 mM NDGA treatment. The treatments began at 3 d after germination and were maintained for 4 d. WT, wild type.(B) and (C) Root length (B) and root elongation (C) measured in the seedlings shown in (A). ND, no significant difference; WT, wild type.

2116 The Plant Cell

ABA and ROS Coordinate in Modulation of Root Elongationby GmSIN1

To better understand the interplay of ABA and ROS in GmSIN1-modulated root elongation, we measured root length in thepresence of ABA and/or ROS biosynthesis inhibitors. To examinetheeffect ofABAdosage, thewild-typeandGmSIN1OEseedlingswere grown at different ABA concentrations in the presence ofFLU to inhibit endogenous ABA synthesis. Consistent with theresults shown in Figures 5F and 5G, the root length of the wild-type and GmSIN1 OE seedlings showed no difference with onlyFLU treatment, but they both showed inverted U curves with therise in ABA concentration andGmSIN1 OE seedlings had longerroots than thewild type at 100 to 10,000 nMABA (Figure 6A). Theoptimal concentration of ABA for root growth seemed to be 100nM in both GmSIN1 OE and control seedlings (Figure 6A).However, when sufficient DPI was added to inhibit NOX activity,the effect of ABA dosage on root elongation was completelysuppressed and the root length difference between the wild-type and GmSIN1 OE seedlings disappeared (Figure 6A). Whenthe root growth assay was performed with DPI treatment anddifferent concentrations of exogenous H2O2 in the wild-typeand GmSIN1 OE seedlings with or without FLU treatment,similar phenotypes to that in ABA treatment were observed(Figure 6B).

Tobetter understand the effect ofABAandH2O2dosageon rootelongation, we treated the wild-type and OE plants with differentconcentrations of ABA or H2O2 without any inhibitor. Low con-centrations of ABA or H2O2 promoted root growth in the wild-typeseedlings and high concentrations of ABA inhibited root growth.By contrast, inOE plants, all concentrations of exogenous ABA orH2O2 inhibited root growth (Supplemental Figures 12A and 12B).This suggested that the ABA andH2O2 levels are closer to optimalinOEplants than in thewild-typeplants; therefore, anyexogenousABA or H2O2 exceeds the optimal level and inhibits growth. InArabidopsis, a similar dosage effect was observed (SupplementalFigures 12C and 12D). Interestingly, an additive effect of lowconcentrations of ABA and H2O2 on root elongation was found inArabidopsis (Supplemental Figure 12E). These results show thatboth ABA and H2O2 have dosage effects on root growth, and theyboth are required to maintain optimal root growth. Additionally,a lowcombinedconcentrationofABAandH2O2mightbebetter forroot growth. Besides the root elongation, seed germinationalso required an optimal ABA level in soybean (SupplementalFigure 12F).

To reveal the details of how ABA and ROS act together in thisprocess, the wild-type andGmSIN1OE seedlings were cultivatedwithmock orNDGA treatment and theO2$

2content, NOXactivity,and transcript levels of GmRbohB genes were tested. NDGAtreatment completely suppressed the increase of O2$

2 and NOXactivity inGmSIN1 OE seedlings in comparison with the wild typebutpromoted the transcriptionofGmRbohBs inboth thewild-typeand GmSIN1 OE seedlings (Figures 6C to 6E). These resultssuggest that ABA is required for GmSIN1 to promote H2O2 pro-duction and acts downstream of transcription of GmRbohBs.Furthermore, DPI treatment suppressed the increase of ABA andNCED activity in GmSIN1 OE seedlings in comparison with thewild typebutpromoted the transcriptionofGmNCED3s inboth thewild-typeandGmSIN1OE seedlings (Figures6F to6H). Therefore,ROS are also required forGmSIN1 to promote ABA accumulationand act downstream of transcription of GmNCED3s.

GmSIN1 Binds to a Unique Motif That Differs from the CoreNAC Binding Motif

The 4-bp core sequence of the NACbindingmotif is CACG (Olsenet al., 2005a); in addition, each NAC transcription factor likely hasaspecificanddistinctbindingmotif (Olsenetal., 2005a;ZhangandGan, 2012; De Clercq et al., 2013). To identify the specific bindingmotif for GmSIN1, we used the Multiple Em for Motif Elicitation(MEME) motif discovery tool (http://meme-suite.org) and our RNA-seq data. We identified a 9-bp consensus sequence, CCTCCACCC,which we named SIN1BM, in 30 of the top 34 genes that wereupregulated by both GmSIN1 and NaCl treatment (Table 2;Supplemental Data Set 4).To determine the key bases in this candidate sequence, we

performed point mutation analysis of SIN1BM and testedGmSIN1 binding to the mutated sequences by Firefly luciferase(LUC) assays. Based on the SIN1BM sequence (CCTCCACCC;Figure 7A), we prepared a series of single-nucleotide–substitutedsequences and fused them to a DNA fragment of the TUBULINBETA CHAIN2 (TUB2) promoter, which does not containa SIN1BM. We first confirmed that GmSIN1 does not bind to theTUB2 promoter (Figure 7B, negative control). Next, we found thatpoint mutations in the second nucleotide (C) and fourth to ninthnucleotides (CCACCC) of the SIN1BM significantly decreasedGmSIN1 binding, and mutations of the first (C) and third (T) nu-cleotide did not significantly reduce the binding (Figure 7B). Thissuggests that the C (second) andCCACCC (fourth to ninth) bases

Figure 5. (continued).

(D)Phenotypeof 7-d-oldGmSIN1OE transgenicplants comparedwithWei6823undermock,100mMDPI, or 50mMNDGAtreatment. The treatmentsbeganat 3 d after germination and were maintained for 4 d. WT, wild type.(E) Root length of seedlings from (D). All data are given as means6 SE (n5 20).OE-1,OE-2, andOE-3 areGmSIN1 OE transgenic lines. The roots of eachgenotypewerecollected fromseedlingsgrown in10plasticgrowthbagswith thesame treatment. Eachbag included threeseedlingseach fromthewild type(WT) and the three transgenic lines. The data shown are a representative result from several independent experiments. ND, no significant difference.(F) Expression ofGmSIN1 analyzed using RT-qPCR in response to 150mMNaCl, 1mMH2O2, and 100 mMABA treatment. FLU (50 nM) or 100 mMDPI wassupplemented or not with NaCl, ABA, or H2O2 in the roots of 6-d-old seedlings of cv Shengdou No. 9.(G) Expression of GmSIN1 analyzed using RT-qPCR in response to 150 mM NaCl for 48 h in soybean leaves transiently transformed with empty vectorpB7GWIWG2(II),p35Spro:GmNCED3-RNAi, orp35Spro:GmRbohB-RNAi. Data aremeans6 SE (n53). Significant differencesbetween samples labeledwithdifferent Roman (a, b, c) orGreek letters (a,b, g) were determinedby one-wayANOVAandTukey’s test, P < 0.05. Thedetails of the samplingprocedures arepresented in the “Methods.”

GmSIN1 in Root Growth and Salt Tolerance 2117

Figure 6. Interaction of ABA and ROS in GmSIN1-Modulated Root Elongation.

(A) Root length of 6-d-old seedlings of GmSIN1 OE-1 transgenic and the wild-type (WT) seedlings grown in the presence of 50 nM FLU and differentconcentrations of ABA with or without 100 mM DPI.(B) Root length of 6-d-old seedlings ofGmSIN1 OE-1 transgenic and the wild-type (WT) seedlings grown with 100 mMDPI and different concentrationsofH2O2withorwithout 50 nMFLU.The treatments beganat 2 dafter germinationandweremaintained for 4d.Data aremeans6 SE (n520seedlings). Therootsof each treatmentwerecollected fromseedlingsgrown inplastic growthbagswith the same treatment. Eachbag included fiveseedlingseachofWTand OE-1. The data shown are a representative result of several independent experiments.(C)NBT staining reveals the superoxide content of the root tips of 6-d-oldGmSIN1OE-1 and thewild-type (WT) seedlings exposed to either 0 mM (mock) or50 mMNDGA. The treatments began at 2 d after germination and were maintained for 4 d. The staining intensity reflects the concentration of superoxide.Bar 5 1 mm.(D)NOX activity in the roots of theGmSIN1OE-1 and thewild-type (WT) plants as in (C). Data aremeans6 SE (n5 5 biological replicates). FW, freshweight.(E) to (G) Transcript abundance of GmRbohB-1 and GmRbohB-2 (E), GmNCED3-1 and GmNCED3-2 (H). Data obtained by RT-qPCR. The roots fromseedlingswith the same treatments as in (C)and (F)wereused forRNApreparation. (F)ABAcontents of the rootsof 6-d-oldGmSIN1OE-1and thewild-type(WT) seedlingsexposed toeither 0mM(mock)or100mMDPI.The treatmentsbeganat2daftergerminationandweremaintained for4d.Errorbarsdenote theSE (n5 3 biological replicates). (G)NECD activity in the roots of theGmSIN1OE andWTplantswith the same treatment as in (F). Data aremeans6 SE (n5 5biological replicates). Asterisks denote significant differencesbetween themutated promoter and theWTpromoter (t test, *P < 0.05, ***P < 0.001). FW, freshweight; ND, no significant difference.

2118 The Plant Cell

are thecoresequenceof theSIN1BMandareessential for theDNAbinding of GmSIN1.

GmSIN1 Binds Directly to the SIN1BM in the Promoters ofGenes Associated with ABA Synthesis and ROS Generation

To investigate whether genes involved in ABA synthesis and ROSgeneration are direct targets of GmSIN1, we looked for candidateSIN1BMs in their promoter regions using FIMO (http://meme-suite.org/tools/fimo). We tested the promoters of GmNCED3-1,GmNCED3-2,GmRbohB-1, andGmRbohB-2 (Supplemental DataSet 5) and found one or two SIN1BMs in each of the promotersof these four genes. For GmRbohB genes, the SIN1BM is locatedin the 59 untranslated region (Figure 8A). To examine whetherGmSIN1 can bind to these sites in the promoters, we performedelectrophoretic mobility shift assays (EMSAs) and LUC assays.

The full-lengthGmSIN1was fused to glutathione transferase (GST)and used for theEMSA, andDNAprobeswere biotin-labeled 50-bppromoter fragments of each gene that contains SIN1BM, with theSIN1BM in the center (probe sequences are listed in SupplementalTable3).GmSIN1couldstronglybindtotheSIN1BMinthepromotersofGmNCED3-1,GmNCED3-2,GmRbohB-1, and GmRbohB-2, andthe binding was efficiently competed off by the unlabeled wild-type probes, but not by unlabeled, SIN1BM-mutated probes(Mutant, Figures 8B), indicating that the binding is specific. TheLUCassayused thesamemethodasused inFigure8B. It revealedthatGmSIN1bound to theSIN1BMs in thepromoters of these fourgenes (Figure 8C).An RT-qPCR–based chromatin immunoprecipitation (ChIP)

assaywasalsodeployed tomonitor thebindingaffinityofGmSIN1to the various gene promoters in 2-week-old Shengdou No. 9seedlings exposed to either 0 or 150mMNaCl for 12 h. Thequality

Table 2. SIN1BMs in Promoters of GmSIN1 and Salt Stress Co-Upregulated Genes

Gene Locus P-Value Consensus MotifHomologs inArabidopsis Annotations

Glyma.14G034200 8.00E208 TCTTTAGTCA CCGCCACCC AACCAGGACA AT5G19420.1 Regulator of chromosome condensation family withFYVE zinc finger domain

Glyma.13G333200 8.00E208 TTAATGAATT CCTCCCCCC CCCCCAAATG AT3G46130.2 Myb domain protein 48Glyma.10G207000 2.08E207 CAAATCACCC CCTCCACCC TACCACTACT –

Glyma.12G230300 4.29E207 CCTTCGGATT CCGCCACCGCCATAACGCC

AT1G16916.1

Glyma.01G240100 5.31E207 AGCTACAGTA CCGCCCACCAGATCGGGAG

AT4G21750.2 MERISTEM LAYER1

Glyma.16G095700 8.34E207 GTTTTCTACA CCGCCACCA TTAAACAACA AT4G26470.1 Calcium-binding EF-hand family proteinGlyma.16G005600 8.34E207 GTCCATGTTG CCGCCACCA TTCGTCGTTA AT1G77320.1 MEIOSIS DEFECTIVE1Glyma.07G196700 9.28E207 GCTAGAAACT CCACCCCCA ACAACGATCT AT4G30930.1 NUCLEAR FUSION DEFECTIVE1Glyma.11G213200 1.81E206 CTGAAAATAC CCACCCACC TAACCTTACA AT2G01990.1Glyma.10G294100 1.81E206 AGGGAGAGTT GCTCCACCC TTTTCCCTCT AT1G04880.1 HMG box protein with ARID/BRIGHT DNA-binding

domainGlyma.19G003700 2.02E206 GATTCCATCA CCGCCACGC GCGCCATTAA AT3G27570.1 Sucrose/ferredoxin-like family proteinGlyma.18G136900 2.58E206 ACCACATCAA CCACCACCA CAACCATTAC AT1G23860.3 RS-containing zinc finger protein21Glyma.09G010500 3.33E206 GTGCTCTACG CCTCTACCC TTTAGAATCGGlyma.05G016700 4.28E206 GGAAGGCATA CTTCCCCCC TAACCCTCTA AT1G73980.1 Phosphoribulokinase/uridine kinase familyGlyma.14G001500 6.20E206 TCATCTGCGC ACACCACCC TCAACCTCTA AT4G38350.1 ATNPC1-2Glyma.02G260300 6.20E-06 AGACACATAT ACACCACCC ACGTGGTTTT AT3G56200.1 Transmembrane amino acid transporter family proteinGlyma.11G199700 6.88E206 TATTGGTAAT CTTCCACCC CTTTCTTAAC AT5G58300.2 Leucine-rich repeat protein kinase family proteinGlyma.06G098900 9.83E206 TTCTTTTGTT CCTCCCCTC CTTTGGAAAA AT1G12680.1 Phosphoenolpyruvate carboxylase-related kinase 2Glyma.17G109200 1.09E205 CAAAACAGAC CCACTCCCA

CGGGTCAAGAAT5G20300.2 TOC90

Glyma.05G014400 1.09E205 AAGACCTTCC CCACTCCCA CTTCGTGTTA AT1G48950.1 C3HC zinc finger-likeGlyma.10G221500 1.35E205 ATGATATTTT CCTCTCACC TCCTGAGTCC AT1G22770.1 GIGANTEAGlyma.13G149600 1.46E205 GGTAAAAAAT CCACCCCGA ATCCCTACTC AT3G53000.1 PHLOEM PROTEIN2-A15Glyma.16G034400 1.94E205 GCATTCAACT CCTTCACCC AACCTATTGT AT1G29320.1 Transducin/WD40 repeat-like superfamily proteinGlyma.05G230700 2.22E205 TGAAAGGTCT CCAACACCC CAGAGAATGG AT5G21222.1 Protein kinase family proteinGlyma.09G031100 2.78E205 TCGCAGCCGT CCACTAACC GTGATTTCCC AT3G06480.1 DEAD box RNA helicase family proteinGlyma.15G248200 2.96E205 CATTACTTAA GCTCCCACG CATTTTTTAA AT1G17110.1 UBP15Glyma.10G215300 2.96E205 GGCTGGAATT GCTCTCCCG AACCCAATTC AT1G22700.2 Tetratricopeptide repeat (TPR)-like superfamily proteinGlyma.05G135900 2.96E205 AGCCAGGAAG GCGCCAACG

CGAATCAAAAAT2G02180.1 TOBAMOVIRUS MULTIPLICATION PROTEIN3

Glyma.15G099100 4.70E205 AAAATGAATT ATGCCACCC ATTAATAATT AT5G18760.1 RING/U-box superfamily protein

ATNPC, Arabidopsis NIEMANN-PICK DISEASE TYPE C1-2; C3HC, C3HC type zinc finger protein; DEAD, DEAD box helicase domain; EF, EF handdomain; FYVE, FYVE zinc finger domain; HMG, HMG, High mobility group box domain; Myb, myb DNA binding domain; RS, arginine/ serine-richdomain; UBP, ubiquitin C-terminal hydrolases; TOC, translocon at the outer chloroplast membrane; WD40, short ;40 amino acid motifs, oftenterminating in a Trp-Asp (W-D) dipeptide. Blank cells or dashes indicate no data.

GmSIN1 in Root Growth and Salt Tolerance 2119

of GmSIN1 antibody was confirmed by immunoblot in GmSIN1transgenic Arabidopsis and soybean and the correspondingwild-type plants (Supplemental Figure 2). Fragments of theGmNCED3and GmRbohB promoters near (b) or far away (a) from SIN1BMs(>1000 bp) were amplified by PCR (Figure 8D). In the ChIP ex-periment, the “b” fragments were enriched much more than “a”fragmentsundersalt stress,but theywereenriched tosimilarextentsunder control conditions (Figure 8D), indicating that GmSIN1 canbind these promoters in vivo via the SIN1BM under salinity stress.

To further confirm that GmSIN1 regulates GmNCED3s andGmRbohBs in vivo, we examined whether GmSIN1 can directlyregulate the transcription of these genes via the SIN1BMs usinga protoplast transient assay system (Figures 8E and 8F). Thewild-typeandSIN1BM-mutatedpromoters of thesegeneswere clonedinto a reporter vector (pGreen II 0800-LUC) as a transcriptionalfusion with the LUC reporter gene, and the effect of GmSIN1 ontranscriptionwasassessedbychanges inLUCactivity (Figure8E).The reporter constructs or control construct were each co-transfected with the effector construct 35Spro:GmSIN1 into eco-type Columbia-0 (Col-0) protoplasts for a gene transcriptionactivity assay. Overexpression of GmSIN1 in the protoplastscaused a significant increase of LUC expression driven by thewild-typepromotersofGmNCED3-1,GmNCED3-2,GmRbohB-1,and GmRbohB-2, relative to the expression driven by theSIN1BM-mutated promoters (Figure 8F). These results suggestthat GmSIN1 regulates transcription of these genes throughbinding to the SIN1BM in their promoters. Thus, GmSIN1 directlytargets the key ABA biosynthesis and ROS generation genesGmNCED3-1, GmNCED3-2, GmRbohB-1, and GmRbohB-2 toregulate their expression, thereby modulating the root elongationin response to salt stress in soybean.

DISCUSSION

Finding the balance between environmental stress tolerance andplant growth is an emerging, important research topic (Deng et al.,2017; Xu et al., 2017;Wang et al., 2018). Here, the overexpression ofGmSIN1 was shown to promote plant growth, whether or not theenvironment has high salinity. The underlying mechanism involvesaGmSIN1/GmNCED3s/GmRbohBs feed-forward loop that rapidlyamplifies the initial stress signal, thereby raising the ABA andROS contents of the soybean root. This observation not onlyupdates our understanding of the regulatory network involvedin the early salinity stress response but also suggests a novelgenetic engineering-based strategy to maintain crop productivityunder environmentally challenging growth conditions.

The Unique Role of GmSIN1 in Root Elongation and SaltTolerance in Soybean Differs from Its Homologs inOther Species

NAC transcription factors associate with abiotic stress tolerance(Lu et al., 2012; Mao et al., 2012, 2015; Puranik et al., 2012; Huangetal., 2015;Yanget al., 2015a).However, fewNACssimultaneouslyimprove abiotic stress tolerance and plant growth asGmSIN1does(Figure1;Table1).Bycontrast,someNACshavenegativeeffectsonplant growth. For example, ectopic expression of MlNAC5 inArabidopsis produced dwarfism and late flowering (Yang et al.,2015a). Moreover, ANAC072 overexpression produced shorterplants, fewer flowers, and reduced seed yield (Fujita et al., 2004),and OsNAC6 overexpression caused growth retardation andlower reproductive yields (Nakashima et al., 2007). Finally,heterologous expression of GmNAC11 in hairy roots causedlower root growth (Hao et al., 2011). This suggests thatGmSIN1plays a unique role in promoting salt tolerance as well as growthin soybean, which might make it valuable for crop improvement.

Figure 7. Identification of the GmSIN1 Binding Motif (SIN1BM).

(A) Using MEME analysis, conserved sequences were identified in thepromoter regions of genes co-upregulated in GmSIN1 OE plants and theNaCl-treated wild-type plants (Table 2). Position weight matrix of SIN1BMshowing the probability of a nucleotide(s) at each position.(B) Interaction of GmSIN1 with the SIN1BM consensus and its substitutedsequences in LUC (Firefly luciferase) assays. The sequences of interestwere fused to a promoter fragment of TUB2 that does not containa SIN1BM. LUC activity and REN (Renilla luciferase) activity were mea-sured. Dots below the sequences indicate the substitution from SIN1BM,which was determined by MEME analysis (shown in [A]). pTUB2pro::LUCand pGreenII 0800-35S-LUC were used for negative control and positivecontrol, respectively. Mean and SE of LUC activity/REN activity were ob-tained from more than four independent transformation experiments.Significant differences between samples labeledwith different letters (a, b,c) were determined by one-way ANOVA and Tukey’s test, P < 0.05.

2120 The Plant Cell

Figure 8. GmSIN1 Upregulates the Expression of GmNCED3s and GmRbohBs by Binding to Their Promoters.

(A) Schematic of SIN1BM binding to the promoters ofGmNCED3-1,GmNCED3-2,GmRbohB-1, andGmRbohB-2. Red lines show the potential SIN1BMbinding site on the promoters, empty boxes indicate the 59 untranslated region (UTR) of the genes, and black blocks indicate the ATG translation initiationsite. Roman numbers indicate the different potential SIN1BMs in the promoters.

GmSIN1 in Root Growth and Salt Tolerance 2121

The GmSIN1 gene isolated from Shengdou No. 9 representsagene locus thatdiffers from its homologsGlyma.12G221500andGlyma.13G279900 in Williams 82. The genes in Arabidopsis withthe closest phylogenetic relationship to GmSIN1 are ANAC019,ANAC055, and ANAC072 (also named RD26). As shown inSupplemental Figure 7A, the products of these six genes sharedstrong peptide sequence similarity. However, the reported ex-perimental data showed that the function of GmSIN1 might bedifferent from its homologs. For example, ANAC019, ANAC055,and ANAC072 are expressed most in stems (Tran et al., 2004;Jensen et al., 2010) rather than in roots, like GmSIN1 (Figures 2Cand 2D). Additionally, their OE transgenic plants were droughttolerant and ABA hypersensitive in root growth and seed germi-nation (Tran et al., 2004; Jensen et al., 2010). By contrast, 35Spro:GmSIN1 transgenic soybeans were salt tolerant and insensitiveto ABA in seed germination (Supplemental Figure 13). Glyma.12G221500 (GmNAC004) and its homologs in the legumeCaragana intermediawere recently reported to improve lateral rootformation and caused insensitivity to ABA in seed germinationwhen overexpressed in transgenic Arabidopsis (Quach et al.,2014; Han et al., 2015). However, improved primary root elon-gation under normal and salt stress conditions in 35Spro:GmSIN1transgenic soybeans was not reported (Figure 1). This suggeststhat GmSIN1 functions differently from its previously reportedhomologs in other plants including legumes.

A Feed-Forward Pathway Involving GmSIN1, GmNCED3s,and GmRbohBs Functions during the Early SaltStress Response

Secondary messengers amplify primary signals, so a fuller un-derstanding of howplants transduce the primary salt stress signalwill facilitate efforts to enhance salt tolerance. Two key secondarymessengers of salinity stress are ABA and ROS, which induceglobal changes in gene expression and metabolism (Finkelstein

and Gibson, 2002). Despite their large effect, how they rapidlyaccumulate and coordinate their effects remains unresolved.NCED3 has been implicated as an important contributor to ABAsynthesis during episodes of moisture deficit or salt stress (Iuchiet al., 2001; Barrero et al., 2006), while RBOH proteins boost ROSproduction in response to salt stress (Xie et al., 2011).NCED3 andRBOH are upregulated by salinity stress (Barrero et al., 2006; Xieet al., 2011), although the identityof theTF(s) involved indriving theaccumulation of either ABA or ROS in the early salinity responseremains unknown. The present experiments have provided evi-dence that the rapid induction of ABA and ROS in soybean inresponse to salt stress was mostly dependent on the presence ofGmSIN1 (Figure 4) and that GmSIN1 promoted the synthesis ofABA and ROS by binding to the GmNCED3 and GmRbohB pro-moters and upregulating the expression of these genes (Figure 4and8).Moreover, ourobservationsshowthatGmSIN1was rapidlyinduced by salt stress or exogenous ABA or ROS, with the in-duction requiring bothABAandROS to be synthesized (Figure 2F,5F and 5G) and that GmSIN1 mediated part of the early tran-scriptional response to salt stress in soybean (Figures 3A to3D). Our major conclusion is that GmSIN1, GmNCED3s, andGmRbohBs work together as a positive feed-forward loop tomediate the rapid accumulation of ABA and ROS, which amplifiesthe salt stress signal (Figure 9).ABA and ROS had been thought of as inhibitors of root elon-

gation (Pilet, 1975) . Recently, increasing evidence shows thatABA and ROS also promote root elongation and maintain rootgrowth. For example, root elongation under conditions of lowwater potential is reduced in ABA-deficient mutants in maize, andelongation can be restored by the addition of ABA (Sharp et al.,2004). Also, ABA biosynthesis and signaling are necessary topromote full growth recovery during salt stress (Geng et al., 2013).The ROSgeneration genesAtRbohC,AtRbohD, andAtRbohF arerequired for root hair tip growth (Foreman et al., 2003) and rootlength in response to ABA (Kwak et al., 2003; Jiao et al., 2013) in

Figure 8. (continued).

(B) EMSA results of GmSIN1 binding with the SIN1BM site in the target gene promoters. (Left to right) Lane 1, free probe (FP; labeled probe with no proteinadded); lane 2, labeled probewithGSTprotein as negative control; lane 3, labeled probewithGmSIN1-GST protein; lanes 4 and 5,GmSIN1-GST binding tothe labeled probe was competed with 503 or 2003 unlabeled wild-type probes (denoted by the empty wedge); lanes 6 and 7, binding was competed withmutant probe sequences (the SIN1BM was mutated; lanes 6 and 7, marked by the filled wedge). The arrows indicate the protein-probe complex. Comp,competitor probe; S-G, GmSIN1-GST fusion protein.(C) Interaction of GmSIN1 with the potential SIN1BMs in LUC assays. The candidate SIN1BM sequences were fused to a promoter fragment of TUB2 thatdoes not contain a SIN1BM. LUC activity and REN activity was measured (see “Methods”). pTUB2mini:LUC and pGreenII 0800-35S-LUC were used fornegative andpositive controls, respectively. I toV indicate thepotential SIN1BMsshown in (A).Meanand SEof LUCactivity/RENactivitywereobtained frommore than four independent transformation experiments.(D)ChIP-qPCR assay showing that GmSIN1 interacts withGmNCED3 andGmRbohB promoters in vivo. Anti-GmSIN1 antibodies were used to precipitatechromatin prepared from1-week-oldShengdouNo. 9 seedlings after 24-hNaCl treatment (150mM)ormock treatment. The fold enrichmentwascalculatedbased on the relative change in anti-GFP samples comparedwith rabbit serum samples (asmock). The “a” and “b” promoter fragments are indicated in (A).Data aremeans6 SE (n53 independent experiments). Significant differencesbetweensamples labeledwithdifferentGreek letters (a,b,g) weredeterminedby one-way ANOVA and Tukey’s test, P < 0.05.(E)Constructs used for transient expression assay. For the constructs, 2-kb fragments of theGmNCED3 andGmRbohB promoters drive LUC expression.Mutant indicates the promoter with the SIN1BM site(s) mutated. The arrow indicates the promoter and the box indicates the coding sequence.(F)Transient assays forGmNCED3 andGmRbohBexpression regulatedbyGmSIN1. Data aremeans6 SE of four independent biological repeats. Asterisksdenote significantdifferencesbetween themutatedpromoter and thewild-typepromoter (t test, *P<0.05, ***P<0.001),n54.1, protoplastscotransformedwith pGreenII-35Spro-LUC and p35Spro:GmSIN1 as positive control. 2, protoplasts cotransformed with pGreenII-0800-LUC and p35Spro:GmSIN1 asnegative control.M, reporter construct containingapromoterwithmutantSIN1BMsite;W, reporter construct containing thewild-type target genepromoterdriving LUC.

2122 The Plant Cell

Arabidopsis. PvRbohB was reported to promote lateral rootelongation in Phaseolus vulgaris (Montiel et al., 2013). Thesefindings suggest that there is an optimal ABA or ROS level for rootelongation and that deviating above or below that level reducesthe ability of the root to elongate. The existence of this dualthreshold has not been directly shown experimentally, and howthe thresholds are regulated remains unknown. In this study, weused biosynthesis inhibitors to eliminate endogenous ABA andROS and supplemented with different concentrations of ABA orROS to assay the effect of their dosage on root growth. ABA andROSbothhavedosageeffects on root elongation, and the optimal

amounts of ABA andROSare essential for root growth in soybean(Figure 6).GmSIN1 also functions in this process, as shown by the fol-

lowing observations. (1) The inhibition of ABA or ROS productioncan completely rescue the increased root growth phenotype ofGmSIN1OE seedlings (Figures 5D, 5E, 6Aand6B), demonstratingthat the effect of GmSIN1 in promoting root growth under bothoptimal and salt stress conditions was mostly dependent on theABAandROSpathway. (2) Overexpression ofGmSIN1 altered thesensitivity to ABA andROS in root elongation (Figures 6A and 6B).This demonstrated that GmSIN1 plays an important role in me-diating ABA- and ROS-dependent root elongation. It also sug-gested thatGmSIN1 improved root elongation by promoting ABAand ROS levels and modulating their sensitivity.The relationship between ABA and ROS in root growth and

salinity tolerance is another issue that needs to be clarified.Generally, ROS act downstream of ABA signals in multiple plantspecies and tissues (Zhang, 2014). However, our results showedthat ROS cannot regulate root elongation without ABA (Figure 6B)and the GmSIN1-promoted ROS accumulation required ABAbiosynthesis (Figures 6C and 6D), and vice versa; i.e., GmSIN1-promoted ABA accumulation required ROS production (Figures6A and 6F). This indicates that in root growth in soybean, ABA andROS have an interdependent relationship, not a simple linearrelationship as previously thought.In the mechanism of ABA-regulated ROS production, RbohF is

phosphorylated and activated by OST1 (Sirichandra et al., 2009)and SNF1-related protein kinase 2 (Umezawa et al., 2013), twopositive regulators of ABA signaling. Consistent with this, ourresults showed that the ABA-dependent increase of ROS accu-mulation in GmSIN1 OE soybean relied on NOX activity ratherthan transcriptional regulation of GmRbohB genes (Figures 6Dand 6E). Notably, we found the increase of ABA level and NCEDactivity caused by GmSIN1 overexpression was impaired by DPItreatment (Figures 6G and 6H) and this effect occurred down-stream of GmNCED3s transcription. This indicated a positiveeffect of ROS on ABA biosynthesis, which has not been shown inprevious studies.Based on the full set of experimental evidence, we propose

aworkingmodel to illustrate themechanismunderlying theGmSIN1/GmNCED3/GmRbohB transduction and amplification of the primarysalt stress signal, using ABA and ROS as secondary signals. BothABA and ROS were required to regulate root growth, and an ap-propriate level of ABA and ROS is optimal for root growth (Figure 9).

GmSIN1 Binds to a cis-Acting Element to RegulateGene Transcription

Plant genomes encode many NAC TFs, with 226 in soybean (Leet al., 2011) and 106 in Arabidopsis (Riechmann et al., 2000).Identifying their binding motifs will help elucidate the functionalspecificity of each NAC TF. So far, the direct binding motifs of 25NACs have been revealed (Supplemental Table 1) and found to bediverse. However, most contain the CGT[GA] sequence (Olsenetal.,2005a;Yabutaetal., 2010;Balazadehetal., 2011;Yangetal.,2011; Lee et al., 2012; Wu et al., 2012; Zhang and Gan, 2012).These results suggest that CGT[GA] may be the core bindingsequence for most NAC proteins, although NAC binding motifs

Figure 9. Model of GmSIN1-Modulated Root Elongation under SaltStress.

Salinity or other abiotic stresses induce the expression ofGmSIN1, whichpromotes the transcription of the ABA biosynthesis geneGmNCED3s andthe ROS generation gene GmRbohBs and thus induces the accumulationofABAandROS.At thesame time,ABAandROSpromote the transcriptionof GmSIN1. GmSIN1, GmNCED3s, and GmRbohBs work as a positivefeedback loop to mediate the rapid accumulation of ABA and ROS inresponse to salt stress (and maybe other abiotic stresses as well). Thesefactors work interdependently to establish optimal ABA and ROS levelsand thus maintain root elongation under salt stress. The arrows anddashed arrows in the gray area indicate direct and indirect regulation,respectively.

GmSIN1 in Root Growth and Salt Tolerance 2123

not containing the core sequence have also been identified. Thespecific DNA binding property of each NAC may contribute to itsfunctional specificity. For example, ANAC016 andANAC017 haveclose phylogenetic relationships but diverse functions: ANAC016regulates drought tolerance and leaf senescence (Kimet al., 2013;Sakuraba et al., 2015), while ANAC017 regulates mitochondrialretrograde signaling (Ng et al., 2013). Their functional differencesmay be due to their different binding motifs and target genes(Sakuraba et al., 2015).

In this study, we used RNA-seq analysis, the MEME motifdiscovery program, and mutation analysis to identify SIN1BM,[CA][CT][TAG]CC[AC]CC[AGC] (Figure 7). Notably, SIN1BM doesnot harbor the CGT[GA] sequence (Figure 7) and differs from thepreviously identified binding motifs of NAC TFs (SupplementalTable 1). GmSIN1 was further demonstrated to bind to theSIN1BMs in the promoters of GmNCED3s and GmRbohBs andupregulate their transcription (Figure 4A; Figure 8). GmSIN1 hasa close phylogenetic relationship and sequence similarity toANAC019/055/072 (Supplemental Figures 9 and 10), which bindto the core DNA sequence CGT[G/A] (Tran et al., 2004; Jensenet al., 2010). Using EMSA, we found that GmSIN1 could also bindto CGT[GA] as did its homologs in Arabidopsis (SupplementalFigure 14; Tran et al., 2004). This suggested that the bindingmotifof this specific NAC TF was not unique and the binding motifs ofNAC TFs are more diverse than had been previously shown. Thisdivergence likely provides diversity in regulation of their targetgenesandgives theplantmoreflexibility in its response toexternalor internal signals.

Overall, the present study has revealed the relationship betweenGmSIN1, GmNCED3, and GmRbohB, which constitute a feed-forward loop responsible for the rapid amplification of the saltstress signal, andhas shownhowGmSIN1upregulatesGmNCED3and GmRbohB by directly binding to a novel motif in their pro-moters. Notably, the process was shown to require ABA and ROS,which suggests that the combinedmanipulation of tissue ABA andROS contents could represent a viable strategy for improving salttolerance. GmSIN1 is therefore held to be a promising potentialtarget for genetic intervention aimed at raising the yield of soybeanin both non saline and saline environments.

METHODS

Plant Materials and Growth Conditions

The soybean (Glycinemax) cv Shengdou No. 9 and cvWei6823 were usedfor gene cloning and phenotypic assays, respectively. For germination,soybean seeds were placed on moistened filter paper for 2 d at 20°C.Seedlings of uniform size were transferred to a 16-h photoperiod regime(light/dark temperature, 28°C/20°C) under 800 mmol m22 s21 illumination(fluorescent lamp) and a relative humidity of 60% and were grown hy-droponically in half-strength Hoagland solution (for RNA preparation as-say) or were grown in plastic root growth bags soaked with water (forphenotype assay). In root growth phenotype assays, to minimize positioneffects, several parallel bags were used for each treatment, and all gen-otypes were included in each bag in the same number. Among differentbags for the same treatment, the materials were placed in different se-quences to avoid the position effects. Abiotic stress was applied to soy-bean seedlings by the addition of 100 or 150 mM NaCl, 100 mM ABA, or1 mM H2O2 to the hydroponic solution.

To test the agronomic traits of field-grown soybeans, seeds wereplanted using a randomized complete block design with six repetitions.Eachplot included four rows in randomarrangementwith thewild-type andthreeOE lines and the six plotswere randomly distributed in the field. Everygenotype had the same number of seeds planted in every row within theplot. The spacingbetween rowswas0.5m, theplot lengthwas2m, and thespacing between plants was 5 cm. The seeds were planted at the end ofMay and harvested at the beginning of October in north China. The soilsalinity is defined as theweight of total soluble salt per 100gof dry soil. Thesoil from;20cmto thesurface ineachplotwascollected forsalinity assaysat ;1 month before sowing. The average salinity was used as the fieldsalinity.

Phylogenetic Analysis

The ClustalW-based alignment of GmSIN1 and other NAC protein poly-peptide sequences used a gap open penalty of 10 and a gap extensionpenalty of 0.2, as implemented within MEGA6 software (Tamura et al.,2013). The resulting phylogenetic tree was derived by the same softwareand used the neighbor-joining method based on the Jones-Taylor-Thornton amino acid substitution model. The Glyma.01G051300.1 se-quence was adopted as the out-group in the phylogenetic analysis, whichencodes a NAC family protein. In total, 1000 bootstrap replicates wereincluded to allow for the assigning of confidence levels to each node. Thesequences used in phylogenetic analysis are listed in Supplemental DataSet 2.

Generation of Transgenic Soybean Plants

To generate the 35Spro:GmSIN1 construct for overexpression in soybean,the GmSIN1 coding DNA sequence (CDS) was amplified with primersGmSIN1-OE-F andGmSIN1-OE-R, and the PCR product was then ligatedinto the Gateway pDONR221 vector (Invitrogen) via a BP recombinationreaction and then were transferred into the pB2GW7 binary vector (underthe control of the 35S promoter; Plant Systems Biology [PSB], GhentUniversity, Belgium) via an LR recombination reaction. The RNAi constructwas designed to target themiddle of theGmSIN1CDS. A 402-bp fragmentwas amplified using primers GmSIN1-RNAi-F and GmSIN1-RNAi-R andinserted in reverse orientation into the binary vector pB7GWIWG2(II) underthe control of the 35S promoter (PSB) to knock downGmSIN1. The binaryplasmids were transferred into the Agrobacterium tumefaciens strainGV3101 using the freeze-thawmethod. Soybean plants were transformedfollowing the protocol described previously (Cui et al., 2013). DNA gelblotting was performed following the manufacturer’s manual (DIG HighPrime DNA Labeling and Detection Starter Kit II, Roche). A DNA fragmentfrom the35Spromoter regionpresent inpB2GW7andpB7GWIWG2(II)wasamplified with primers 35S-F and 35S-R and then labeled as probe. Theprimer sequences used are listed in Supplemental Table 4.

cDNA Synthesis and RT-qPCR

The cDNA synthesis, RT-qPCR, and data analysis were conducted asdescribed previously by Li et al. (2016). Gene-specific primers (sequencesgiven in Supplemental Table 4) were designed using Beacon Designer v7.90 (http://www.premierbiosoft.com). GmTUB was used as the internalreference gene for profiling across the plant (Song et al., 2014),Gm60S forH2O2 treatments, and GmELF1b for the salinity and ABA treatments (Leet al., 2012). Tissues from three to five seedlings under the same treatmentwerepooled forRNAextractionasonebiological replicate. Threebiologicalreplicates from independent experiments were included for each treat-ment. For qPCR, each sample was amplified in three parallel reactions astechnical replicates. The average cycle threshold (Ct) value of threetechnical replicates was assigned as the final Ct value of each biological

2124 The Plant Cell

replicate. The relative gene expression level was calculated using the2–DDCT method (Livak and Schmittgen, 2001).

Subcellular Localization of GmSIN1 Protein

The GmSIN1 CDS was fused to GFP and inserted into the pART27-GFPplant expression vector (kindly provided by Shuqing Cao, Hefei Universityof Technology, Hefei, China), containing the GFP coding sequence underthe control of the 35S promoter. This generated the p35Spro:GmSIN1-GFPconstruct. The Arabidopsis protoplast transformation and GFP signalobservation were conducted as described previously (Li et al., 2016). Thep35Spro:GFP (Li et al., 2011) transgenic protoplasts were used as locali-zation controls for expression in the cytoplasm/nucleus. The pMDC32-1ABES1n-mCherrywasusedasan indicatorof thenucleus (Liangetal., 2015).

Transcriptional Activation Activity Assay

The GmSIN1 CDS in Gateway pDONR221 vector was transferred into thepDEST32 vector (Invitrogen) via an LR recombination reaction, and thetranscriptional activation activity assay was performed as describedpreviously (Li et al., 2016).

Transient Transformation of Soybean Leaves

For RNAi constructs, a 252-bp cDNA fragment of GmNCED3-1 (Glyma.15G250100) and a 218-bp fragment of GmRbohB-3 (Glyma.19G233900.1)were PCR amplified using primers provided in Supplemental Table 4 and in-serted into the binary vector pB7GWIWG2(II) (under the control of the 35Spromoter, PSB). These constructs were introduced into the A. tumefaciensstrainGV3101.ThestrainscarryingpB7GWIWG2(II) (asvectorcontrol),p35Spro:GmNCED3-RNAi, or p35Spro:GmRbohB-RNAi, together with the strain car-rying p35Spro:p19, were co-infiltrated into leaf epidermal cells of 2-week oldsoybean plants (Shengdou No. 9), following a protocol described pre-viously (Hanano and Goto, 2011). After infiltration for 24 h, the seedlingswere subject to 150 mM NaCl treatment or mock treatment for another48 h. The transformed leaveswere used for RNAextraction andRT-qPCRassay. Three transformed leaves from different seedlings were pooled asone biological repeat, and three biological repeatswere included for eachtransformation.

RNA-Seq Assay

Total RNAwas extracted frommock-treated andNaCl-treated 2-week-oldseedlings (Wei6823 and GmSIN1 OE-1) using the RNeasy Plant Mini kit(Qiagen) according to the manufacturer’s instructions. The total RNA fromrootsand leaveswas isolatedseparatelyandcombined inanequalmixture.A pool of tissues from at least three seedlings was considered a biologicalreplicate. ThemRNAsequencing librarieswere constructedwith barcodesusing the TrueSeq RNA Sample Preparation kit (Illumina). Two biologicalreplicateswere sequencedon an IlluminaHiSeq 2000 systembyBGI-Tech(Shenzhen, China), resulting in 16 to 18 million 49-bp single-end reads persample. We used BWA (Li and Durbin, 2009) to map clean reads to thesoybeangenome (GlycinemaxWm82.a2.v1 inPhytozomev11.0database;https://phytozome.jgi.doe.gov/). DEG identification was based on theNoiseq method (Tarazona et al., 2011) with fold change $ 2 and divergeprobability $ 0.7. The RNA-seq data sets used in this study have beendeposited at Gene Expression Omnibus under accession numberGSE93322 (secure token for reviewers: wpitymcihbcjbqx). Hierarchicalclustering was performed using Cluster v3.0 software (Human GenomeCenter, University of Tokyo) and visualized by Java TreeView v1.1.1(Saldanha, 2004).

KEGG Pathway and GO Function Enrichment Analysis

KyotoEncyclopediaofGenesandGenomes (KEGG;Kanehisaet al., 2008),themajor public pathway-related database, was used to perform pathwayenrichment analysis of DEGs. This analysis identifies significantly enrichedmetabolic pathways or signal transduction pathways in DEGs comparedwith the whole-genome background. The calculated P-value was sub-jected to Bonferroni correction (Abdi, 2007), taking a corrected P-value#

0.05 as a threshold for significance.GO termenrichment analysis of the gene sets of interest was performed

to identify enrichedGOterms.ThecalculationofP-valuewasconductedbythe samemethod as that in KEGGpathway enrichment analysis. GO termsfulfilling this condition are defined as significantly enriched GO termsin DEGs.

DAB and NBT Staining

The whole seedlings of 6-d-old (for root stain) or 17-d-old (for leaf stain)soybean seedlings were treated with 0 or 100 mM NaCl in half-strengthHoagland solution for 2 h before staining. The treated 6-d-old wholeseedlingswere stained to see the signals in roots, and the detached leavesfrom 17-d-old plants were stained directly. For DAB staining, the sampleswere submerged in theDABsolution (CSB-K09758AB-2,CUSABIO) for 2hand then in 95% (v/v) ethanol to decolor. For NBT staining, the sampleswere submerged in NBT staining solution (AR-0632, DINGGUO) until thedark blue color appeared (;10 min for roots and 3 h for leaves). Next, thesamples were cleared of chlorophyll with 95% (v/v) ethanol. The photo-graphs were taken with a stereomicroscope (SZX16, Olympus).

Quantification of ABA Content, H2O2 Content, NOX Activity, andNCED Activity

Tissue ABA contents were measured by liquid chromatography–tandemmass spectrometry (LC-MS/MS-8030 Plus triple quadrupole mass spec-trometer, Shimadzu) as follows. In total, 200 mg of liquid nitrogen–frozen fresh sample was ground with a small glass pestle in a 2-mL vial.Following theaddition of 1.0mLof 80% (v/v)methanol, homogenateswerewell mixed in an ultrasonic bath and then kept overnight at 4°C. After beingcentrifugedat15,200g for 10minat4°C, thesupernatantwascollectedandthen vacuumed to dryness in an RCT-60 concentrator (Jouan). Dried extractwasdissolved in200mLofsodiumphosphatesolution (0.1mol/L,pH7.8)andthen passed through a Sep-Pak C18 cartridge (Waters). The cartridge waselutedwith 1500mL of 80% (v/v) methanol, and the eluatewas vacuumed todryness again. After being dissolved in 50mL of 10% (v/v) methanol, 5mL ofthe solution was injected into the LC-MS/MS system. LC was performedusing a 2.1 3 100-mm AcquityUltra Performance Liquid ChromatographEthyleneBridgedHybridcolumn (1.7mm,Waters)withacolumn temperatureof40°C.ThemobilephasecomposedofsolventA (0.02%[v/v]aqueousaceticacid) and solvent B (100% [v/v] acetonitrile) was used in a gradient mode(time/A concentration/B concentration [min/%/%] for 0/90/10, 4/30/70,5/0/100, 6/90/10) at an eluant flow rate of 0.25mL/min. The systemwas setto multiple reaction monitoring mode using electrospray ionization innegative ion mode with operation conditions as follows: nebulizing gasflow of 3 L/min, drying gas flow of 15 L/min, desolvation temperature of250°C, and a heat block temperature of 480°C. Deuterium-labeled ABA(2H6-ABA, Olchemim) was used as an internal standard. For ABA, theionizationconditionspre-bias voltageof19V for quadrupole1and28V forquadrupole 3, collision energy of 10 eV, andmass-to-charge ratio (m/z) of263/153.2 were used. For 2H6-ABA, the ionization conditions pre-biasvoltage of 20 V for quadrupole 1 and 15 V for quadrupole 3, collisionenergy of 11 eV, and m/z of 269/159.2 were used. The sample ABAcontents were calculated according to the calibration curve establishedusing the internal standard of 2H6-ABA.

GmSIN1 in Root Growth and Salt Tolerance 2125

H2O2contentwasdetermined following theprotocol asdescribedbyLiuet al. (2014). For the NOX activity assay, a 100-mg sample of fresh tissuewas snap frozen in liquid nitrogen and powdered and then treated with anNADPH oxidase assay kit (Nanjing Jiancheng Bioengineering Institute),following the manufacturer’s protocol.

TheNCEDactivity wasmeasuredwith the Plant NCEDELISA kit (USCNLife Science) according to the manufacturer’s manual.

Tissues from several seedlings (more than three) under the sametreatmentwere pooled as onebiological replicate. Three ormore biologicalreplicates were included for each assay. For roots, the 1-cm root tips werecollected. For leaves, the fully expanded true leaves were collected.

MEME Analysis

For MEME analysis, sequences corresponding to the promoter region(1500 to 0 from ATG codon) of the upregulated genes (Supplemental DataSet 6) were obtained from Phytozome 11 (https://phytozome.jgi.doe.gov/pz/portal.html). Upstream DNA sequences were submitted to the MEMEprogram and analyzed using the previously described parameters (http://meme.nbcr.net/meme/cgi-bin/meme.cgi; Bailey and Elkan, 1994).

Transient Assays of Gene Transcription

To study whether GmSIN1 can bind to specific motifs or promoters toregulate target gene expression, the pGreen II 0800-LUC vector system(Hellens et al., 2005) was used and the promoters were cloned into thevector to generate reporter constructs, respectively. The primers used toclone the promoters or to generate themutation are listed in SupplementalTable 4. Each reporter construct, together with either 35Spro:GmSIN1construct, was cotransformed into Arabidopsis (Col-0) protoplasts usingthe same method as described in “Subcellular Localization of GmSIN1Protein” for transcription activity assay. The signals of Firefly and RenillaLUC were assayed using the Dual-Luciferase Reporter Assay System(Promega). A transformation of ;2 3 104 protoplasts in one tube wasconsidered one biological replicate. For qPCR, each sample was tested inthreeparallel reactions for LUCactivity as technical replicates. Theaveragerelative LUCactivity value of three technical replicateswas assigned as thefinal valueof eachbiological replicate. Four ormorebiological repeatswereconducted to obtain the final results.

Expression of Recombinant GmSIN1 Protein and EMSA

Full-length GmSIN1 protein fused with GST was expressed in the vectorpGEX-T in the Escherichia coli strain BL21. The protein extraction andEMSA were conducted as described previously (Yu et al., 2016). The ol-igonucleotide probe sequences are listed in Supplemental Table 4.

ChIP-qPCR Assays

ThesyntheticpeptidesofGmSIN1 (CGGGHVGTSVPQK)were injected intorabbits to generate the corresponding polyclonal antibodies by MWBIOTECH (HK) LIMITED. The specificities of anti-GmSIN1 polyclonal an-tibodies were determined by immunoblot analysis using the wild-typesoybeans, 35Spro:GmSIN1 transgenic Arabidopsis, and wild-type Arabi-dopsis (Col-0; Supplemental Figure 2). ChIP was conducted as describedpreviously (Yu et al., 2016). The plants of 2-week-old Shengdou No. 9seedlings were treated with 150 mMNaCl or H2O asmock-treated controlfor 12 h. Briefly, 3 g of whole seedlings was fixed by immersion in 1% (v/v)formaldehyde and then the chromatin was sheared by sonication to a sizerange of;100 to 1000 bp. After centrifugation at 13,000 rpm for 10 min at4°C, the complex was immunoprecipitated with a GmSIN1-specific anti-body at dilution 1:600or rabbit serumprotein (asmock sample). Primers forpromoters of candidate GmSIN1 target genes or negative control genes

were used todetect thecorrespondingpromoters in theChIPproducts andthe primers used in this study are listed in Supplemental Table 4. Theenrichment percentages were calculated based on the relative change inanti-GmSIN1 compared with input samples. The mock samples did notgenerate enough PCR product to detect. The mean and SD are shown forthree independent ChIP experiments, and the significance of differencesbetweenmeanswas assessedwith one-way analysis of variance (ANOVA)and Tukey’s test. Each immunoprecipitation sample with a pool of severalseedlings is considered one biological replicate. Three biological repeatswere conducted.

Statistical Methods

For comparisons between two sample groups, the Student’s t test wasapplied. For multiple comparison, significance analysis was performedwith one-wayANOVA followedby post hoc tests. Tukey’s test or Dunnett’st test were used in the post hoc tests. Two-way ANOVA was conducted totest the interaction between two factors. The statistical analysis wasperformed using IBM SPSS Statistics software version 25 (IBM). Details ofstatistical results are in Supplemental Data Set 1.

Accession Numbers

Sequencedata from this article canbe found in thePhytozomeorGenBankdatabases under the following accession numbers: GmSIN1 (KY661969),GmRbohB-1 (Glyma.10G152200), GmRbohB-2 (Glyma.20G236200),GmNCED3-1 (Glyma.15G250100),GmNCED3-2 (Glyma.08G176300),GmTUB (Glyma.08G014200), Gm60S (Glyma.13G318800); GmELF1b(Glyma.02G276600), AtTUB2 (AT5G62690).

The RNA-seq data sets used in this study have been deposited at theGene Expression Omnibus under accession number GSE93322.

Supplemental Data

Supplemental Figure 1. Identification of GmSIN1 overexpression andRNAi transgenic soybeans.

Supplemental Figure 2. Confirmation of the presence of GmSIN1protein in soybean plants overexpressing GmSIN1.

Supplemental Figure 3. Fixation of the transgene in three soybeanlines overexpressing GmSIN1.

Supplemental Figure 4. Normal root growth in transgenic soybeanharboring an empty vector.

Supplemental Figure 5. The effect of GmSIN1 overexpression on salttolerance.

Supplemental Figure 6. Yield of GmSIN1 OE and the wild-type plantsgrown in fields with different salinity.

Supplemental Figure 7. GmSIN1 and its homologs in Arabidopsis.

Supplemental Figure 8. Subcellular localization of GmSIN1.

Supplemental Figure 9. Expression GmSIN1 and selected ABA- orROS- responsive genes.

Supplemental Figure 10. GmRbohB-1 and GmNCED3-1 overexpres-sion in transiently transformed soybean leaves.

Supplemental Figure 11. FLU inhibits root growth.

Supplemental Figure 12. The effect of ABA and H2O2 on rootelongation in Arabidopsis and soybean and germination in soybean.

Supplemental Figure 13. Germination of 35Spro:GmSIN1 transgenicsoybeans.

2126 The Plant Cell

Supplemental Figure 14. GmSIN1 binds to CGT[AG] sites in theGmRbohB-1 promoter.

Supplemental Table 1. NAC proteins and their binding motifs.

Supplemental Table 2. The design of the RNA-Seq experiment.

Supplemental Table 3. Probe sequences.

Supplemental Table 4. Primer sequences.

Supplemental Data Set 1. Statistical results.

Supplemental Data Set 2. Polypeptide sequence of GmSIN1 and itshomologs in soybean and A. thaliana.

Supplemental Data Set 3. Differentially transcribed genes derivedfrom the RNA-Seq experiment.

Supplemental Data Set 4. The GmSIN1 binding motif predicted byMEME analysis.

Supplemental Data Set 5. Promoter sequences of genes related toABA synthesis or ROS generation.

Supplemental Data Set 6. Promoter sequences used for MEMEanalysis.

ACKNOWLEDGMENTS

This research was financially supported by the National Key Research andDevelopment Program of China (grant 2016YFD0101902), National Trans-genic Project of China (grants 2018ZX08009-14B and 2016ZX08010002-002), the Major Program of Shandong Province Natural Science Founda-tion (grant ZR2018ZC0334), National Natural Science Foundation of China(grants 31770317, 31471515, 31201269, and 30970243), the NationalSpecial Science Research Program of China (grant 2013CB967300),and the National High Technology Research and Development Program“863” (grant 2013AA102602-4).

AUTHOR CONTRIBUTIONS

S.L. and F.X. supervised and designed the experiments; N.W., D.J., andW.Z. performed most of the experiments; S.L., Y.W., Y.Y., M.L., S.Z., J.Y.,W.Z., J.T., L.X., Y.Z., L.W., X.W., and Z.L. performed part of the experi-ments;M.B. analyzedpart of thedata; S.L. analyzed the data andwrote thearticle; and F.X. supervised and assisted with the writing.

Received September 5, 2018; revised May 7, 2019; accepted June 17,2019; published June 21, 2019.

REFERENCES

Abdi, H. (2007). The Bonferroni and Sidák corrections for multiplecomparisons. In Encyclopedia of Measurement and Statistics, N.J.Salkind, ed (Thousand Oaks, CA: Sage).

Almagro, L., Gómez Ros, L.V., Belchi-Navarro, S., Bru, R., RosBarceló, A., and Pedreño, M.A. (2009). Class III peroxidases inplant defence reactions. J. Exp. Bot. 60: 377–390.

Bailey, T.L., and Elkan, C. (1994). Fitting a mixture model by ex-pectation maximization to discover motifs in biopolymers. Proc IntConf Intell Syst Mol Biol 2: 28–36.

Balazadeh, S., Kwasniewski, M., Caldana, C., Mehrnia, M., Zanor,M.I., Xue, G.P., and Mueller-Roeber, B. (2011). ORS1, an H2O2

-responsive NAC transcription factor, controls senescence in Ara-bidopsis thaliana. Mol. Plant 4: 346–360.

Barrero, J.M., Rodríguez, P.L., Quesada, V., Piqueras, P., Ponce,M.R., and Micol, J.L. (2006). Both abscisic acid (ABA)-dependentand ABA-independent pathways govern the induction of NCED3,AAO3 and ABA1 in response to salt stress. Plant Cell Environ. 29:2000–2008.

Chen, M., Wang, Q.Y., Cheng, X.G., Xu, Z.S., Li, L.C., Ye, X.G., Xia,L.Q., and Ma, Y.Z. (2007). GmDREB2, a soybean DRE-bindingtranscription factor, conferred drought and high-salt tolerance intransgenic plants. Biochem. Biophys. Res. Commun. 353: 299–305.

Creelman, R.A., Bell, E., and Mullet, J.E. (1992). Involvement ofa lipoxygenase-like enzyme in abscisic acid biosynthesis. PlantPhysiol. 99: 1258–1260.

Cui, Y., Barampuram, S., Stacey, M.G., Hancock, C.N., Findley, S.,Mathieu, M., Zhang, Z., Parrott, W.A., and Stacey, G. (2013). Tnt1retrotransposon mutagenesis: A tool for soybean functional ge-nomics. Plant Physiol. 161: 36–47.

De Clercq, I., et al. (2013). The membrane-bound NAC transcription factorANAC013 functions in mitochondrial retrograde regulation of the oxi-dative stress response in Arabidopsis. Plant Cell 25: 3472–3490.

Deng, Y., et al. (2017). Epigenetic regulation of antagonistic receptorsconfers rice blast resistance with yield balance. Science 355:962–965.

Fan, W., Xu, J.M., Wu, P., Yang, Z.X., Lou, H.Q., Chen, W.W., Jin,J.F., Zheng, S.J., and Yang, J.L. (2019). Alleviation by abscisic acidof Al toxicity in rice bean is not associated with citrate efflux butdepends on ABI5-mediated signal transduction pathways. J. Integr.Plant Biol. 61: 140–154.

Finkelstein, R.R., and Gibson, S.I. (2002). ABA and sugar interactionsregulating development: Cross-talk or voices in a crowd? Curr.Opin. Plant Biol. 5: 26–32.

Foreman, J., Demidchik, V., Bothwell, J.H., Mylona, P., Miedema,H., Torres, M.A., Linstead, P., Costa, S., Brownlee, C., Jones,J.D., Davies, J.M., and Dolan, L. (2003). Reactive oxygen speciesproduced by NADPH oxidase regulate plant cell growth. Nature422: 442–446.

Fujita, M., Fujita, Y., Maruyama, K., Seki, M., Hiratsu, K., Ohme-Takagi, M., Tran, L.S., Yamaguchi-Shinozaki, K., and Shinozaki,K. (2004). A dehydration-induced NAC protein, RD26, is involved ina novel ABA-dependent stress-signaling pathway. Plant J. 39:863–876.

Fujita, Y., Fujita, M., Satoh, R., Maruyama, K., Parvez, M.M., Seki,M., Hiratsu, K., Ohme-Takagi, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2005). AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance inArabidopsis. Plant Cell 17: 3470–3488.

Furihata, T., Maruyama, K., Fujita, Y., Umezawa, T., Yoshida, R.,Shinozaki, K., and Yamaguchi-Shinozaki, K. (2006). Abscisicacid-dependent multisite phosphorylation regulates the activity ofa transcription activator AREB1. Proc. Natl. Acad. Sci. USA 103:1988–1993.

Geng, Y., Wu, R., Wee, C.W., Xie, F., Wei, X., Chan, P.M., Tham, C.,Duan, L., and Dinneny, J.R. (2013). A spatio-temporal un-derstanding of growth regulation during the salt stress response inArabidopsis. Plant Cell 25: 2132–2154.

Guan, R., et al. (2014). Salinity tolerance in soybean is modulated bynatural variation in GmSALT3. Plant J. 80: 937–950.

Han, X., Feng, Z., Xing, D., Yang, Q., Wang, R., Qi, L., and Li, G. (2015).Two NAC transcription factors from Caragana intermedia altered salttolerance of the transgenic Arabidopsis. BMC Plant Biol. 15: 208.

Hanano, S., and Goto, K. (2011). Arabidopsis TERMINAL FLOWER1is involved in the regulation of flowering time and inflorescence

GmSIN1 in Root Growth and Salt Tolerance 2127

development through transcriptional repression. Plant Cell 23:3172–3184.

Hao, Y.J., et al. (2011). Soybean NAC transcription factors promoteabiotic stress tolerance and lateral root formation in transgenicplants. Plant J. 68: 302–313.

He, J., Duan, Y., Hua, D., Fan, G., Wang, L., Liu, Y., Chen, Z., Han,L., Qu, L.J., and Gong, Z. (2012). DEXH box RNA helicase-mediated mitochondrial reactive oxygen species production inArabidopsis mediates crosstalk between abscisic acid and auxinsignaling. Plant Cell 24: 1815–1833.

Hellens, R.P., Allan, A.C., Friel, E.N., Bolitho, K., Grafton, K.,Templeton, M.D., Karunairetnam, S., Gleave, A.P., and Laing,W.A. (2005). Transient expression vectors for functional genomics,quantification of promoter activity and RNA silencing in plants. PlantMethods 1: 13.

Huang, Q., Wang, Y., Li, B., Chang, J., Chen, M., Li, K., Yang, G.,and He, G. (2015). TaNAC29, a NAC transcription factor fromwheat, enhances salt and drought tolerance in transgenic Arabi-dopsis. BMC Plant Biol. 15: 268.

Iuchi, S., Kobayashi, M., Taji, T., Naramoto, M., Seki, M., Kato, T.,Tabata, S., Kakubari, Y., Yamaguchi-Shinozaki, K., andShinozaki, K. (2001). Regulation of drought tolerance by genemanipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzymein abscisic acid biosynthesis in Arabidopsis. Plant J. 27: 325–333.

Jannat, R., Uraji, M., Morofuji, M., Islam, M.M., Bloom, R.E.,Nakamura, Y., McClung, C.R., Schroeder, J.I., Mori, I.C., andMurata, Y. (2011). Roles of intracellular hydrogen peroxide accu-mulation in abscisic acid signaling in Arabidopsis guard cells.J. Plant Physiol. 168: 1919–1926.

Jensen, M.K., Kjaersgaard, T., Nielsen, M.M., Galberg, P., Petersen, K.,O’Shea, C., and Skriver, K. (2010). The Arabidopsis thaliana NACtranscription factor family: Structure-function relationships and deter-minants of ANAC019 stress signalling. Biochem. J. 426: 183–196.

Jeong, J.S., Kim, Y.S., Baek, K.H., Jung, H., Ha, S.H., Do Choi, Y.,Kim, M., Reuzeau, C., and Kim, J.K. (2010). Root-specific ex-pression of OsNAC10 improves drought tolerance and grain yield inrice under field drought conditions. Plant Physiol. 153: 185–197.

Ji, D.D., Liu, C., Cao, Q.C., and Xiang, F.N. (2011). Comparison ofthe salt tolerance at sprout and seedling in different soybean.Journal of Shandong Polytechnic University 25: 4–7.

Jiang, M., and Zhang, J. (2002). Water stress-induced abscisic acidaccumulation triggers the increased generation of reactive oxygenspecies and up-regulates the activities of antioxidant enzymes inmaize leaves. J. Exp. Bot. 53: 2401–2410.

Jiao, Y., Sun, L., Song, Y., Wang, L., Liu, L., Zhang, L., Liu, B., Li, N.,Miao, C., and Hao, F. (2013). AtrbohD and AtrbohF positivelyregulate abscisic acid-inhibited primary root growth by affectingCa21 signalling and auxin response of roots in Arabidopsis. J. Exp.Bot. 64: 4183–4192.

Kanehisa, M., Araki, M., Goto, S., Hattori, M., Hirakawa, M., Itoh,M., Katayama, T., Kawashima, S., Okuda, S., Tokimatsu, T., andYamanishi, Y. (2008). KEGG for linking genomes to life and theenvironment. Nucleic Acids Res. 36 (Database): D480–D484.

Kim, Y.S., Sakuraba, Y., Han, S.H., Yoo, S.C., and Paek, N.C.(2013). Mutation of the Arabidopsis NAC016 transcription factordelays leaf senescence. Plant Cell Physiol. 54: 1660–1672.

Kurusu, T., Kuchitsu, K., and Tada, Y. (2015). Plant signaling net-works involving Ca(21) and Rboh/Nox-mediated ROS productionunder salinity stress. Front. Plant Sci. 6: 427.

Kwak, J.M., Mori, I.C., Pei, Z.M., Leonhardt, N., Torres, M.A.,Dangl, J.L., Bloom, R.E., Bodde, S., Jones, J.D., and Schroeder,J.I. (2003). NADPH oxidase AtrbohD and AtrbohF genes function

in ROS-dependent ABA signaling in Arabidopsis. EMBO J. 22:2623–2633.

Le, D.T., Nishiyama, R., Watanabe, Y., Mochida, K., Yamaguchi-Shinozaki, K., Shinozaki, K., and Tran, L.S. (2011). Genome-widesurvey and expression analysis of the plant-specific NAC tran-scription factor family in soybean during development and de-hydration stress. DNA Res. 18: 263–276.

Le, D.T., Aldrich, D.L., Valliyodan, B., Watanabe, Y., Ha, C.V.,Nishiyama, R., Guttikonda, S.K., Quach, T.N., Gutierrez-Gonzalez,J.J., Tran, L.S., and Nguyen, H.T. (2012). Evaluation of candidate ref-erence genes for normalization of quantitative RT-PCR in soybean tis-sues under various abiotic stress conditions. PLoS One 7: e46487.

Lee, S., Seo, P.J., Lee, H.J., and Park, C.M. (2012). A NAC tran-scription factor NTL4 promotes reactive oxygen species productionduring drought-induced leaf senescence in Arabidopsis. Plant J. 70:831–844.

Lefebvre, V., North, H., Frey, A., Sotta, B., Seo, M., Okamoto, M.,Nambara, E., and Marion-Poll, A. (2006). Functional analysis ofArabidopsis NCED6 and NCED9 genes indicates that ABA synthe-sized in the endosperm is involved in the induction of seed dor-mancy. Plant J. 45: 309–319.

Li, H., and Durbin, R. (2009). Fast and accurate short read alignmentwith Burrows-Wheeler transform. Bioinformatics 25: 1754–1760.

Li, S., Wang, N., Ji, D., Xue, Z., Yu, Y., Jiang, Y., Liu, J., Liu, Z., andXiang, F. (2016). Evolutionary and functional analysis of membrane-bound NAC transcription factor genes in soybean. Plant Physiol.172: 1804–1820.

Li, X., Wang, X., Yang, Y., Li, R., He, Q., Fang, X., Luu, D.T., Maurel,C., and Lin, J. (2011). Single-molecule analysis of PIP2;1 dynamicsand partitioning reveals multiple modes of Arabidopsis plasmamembrane aquaporin regulation. Plant Cell 23: 3780–3797.

Liang, M., Li, H., Zhou, F., Li, H., Liu, J., Hao, Y., Wang, Y., Zhao, H.,and Han, S. (2015). Subcellular distribution of NTL transcriptionfactors in Arabidopsis thaliana. Traffic 16: 1062–1074.

Lin, F., Ding, H., Wang, J., Zhang, H., Zhang, A., Zhang, Y., Tan, M.,Dong, W., and Jiang, M. (2009). Positive feedback regulation ofmaize NADPH oxidase by mitogen-activated protein kinase cas-cade in abscisic acid signalling. J. Exp. Bot. 60: 3221–3238.

Liu, S., Liu, S., Wang, M., Wei, T., Meng, C., Wang, M., and Xia, G.(2014). A wheat SIMILAR TO RCD-ONE gene enhances seedlinggrowth and abiotic stress resistance by modulating redox homeo-stasis and maintaining genomic integrity. Plant Cell 26: 164–180.

Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative geneexpression data using real-time quantitative PCR and the 2(-deltadelta C(T)) method. Methods 25: 402–408.

Lu, M., Ying, S., Zhang, D.F., Shi, Y.S., Song, Y.C., Wang, T.Y., andLi, Y. (2012). A maize stress-responsive NAC transcription factor,ZmSNAC1, confers enhanced tolerance to dehydration in trans-genic Arabidopsis. Plant Cell Rep. 31: 1701–1711.

Mao, H., Wang, H., Liu, S., Li, Z., Yang, X., Yan, J., Li, J., Tran, L.S., andQin, F. (2015). A transposable element in a NAC gene is associated withdrought tolerance in maize seedlings. Nat. Commun. 6: 8326.

Mao, X., Zhang, H., Qian, X., Li, A., Zhao, G., and Jing, R. (2012).TaNAC2, a NAC-type wheat transcription factor conferring en-hanced multiple abiotic stress tolerances in Arabidopsis. J. Exp.Bot. 63: 2933–2946.

Marino, D., Dunand, C., Puppo, A., and Pauly, N. (2012). A burst ofplant NADPH oxidases. Trends Plant Sci. 17: 9–15.

Mazel, A., Leshem, Y., Tiwari, B.S., and Levine, A. (2004). Inductionof salt and osmotic stress tolerance by overexpression of an intracel-lular vesicle trafficking protein AtRab7 (AtRabG3e). Plant Physiol. 134:118–128.

2128 The Plant Cell

Min, J.H., Chung, J.S., Lee, K.H., and Kim, C.S. (2015). TheCONSTANS-like 4 transcription factor, AtCOL4, positively regulatesabiotic stress tolerance through an abscisic acid-dependent man-ner in Arabidopsis. J. Integr. Plant Biol. 57: 313–324.

Mittler, R. (2017). ROS are good. Trends Plant Sci. 22: 11–19.Mittler, R., and Blumwald, E. (2015). The roles of ROS and ABA in

systemic acquired acclimation. Plant Cell 27: 64–70.Montiel, J., Arthikala, M.K., and Quinto, C. (2013). Phaseolus vul-

garis RbohB functions in lateral root development. Plant Signal.Behav. 8: e22694.

Munns, R., and Tester, M. (2008). Mechanisms of salinity tolerance.Annu. Rev. Plant Biol. 59: 651–681.

Nakashima, K., Tran, L.S., Van Nguyen, D., Fujita, M., Maruyama,K., Todaka, D., Ito, Y., Hayashi, N., Shinozaki, K., andYamaguchi-Shinozaki, K. (2007). Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and bioticstress-responsive gene expression in rice. Plant J. 51: 617–630.

Nambara, E., and Marion-Poll, A. (2005). Abscisic acid biosynthesisand catabolism. Annu. Rev. Plant Biol. 56: 165–185.

Ng, S., et al. (2013). A membrane-bound NAC transcription factor,ANAC017, mediates mitochondrial retrograde signaling in Arabi-dopsis. Plant Cell 25: 3450–3471.

Olsen, A.N., Ernst, H.A., Leggio, L.L., and Skriver, K. (2005a). DNA-binding specificity and molecular functions of NAC transcriptionfactors. Plant Sci. 169: 785–797.

Olsen, A.N., Ernst, H.A., Leggio, L.L., and Skriver, K. (2005b). NACtranscription factors: Structurally distinct, functionally diverse.Trends Plant Sci. 10: 79–87.

Papiernik, S.K., Grieve, C.M., Lesch, S.M., and Yates, S.R. (2005).Effects of salinity, imazethapyr, and chlorimuron application onsoybean growth and yield. Commun. Soil Sci. Plant Anal. 36:951–967.

Perea-Resa, C., Carrasco-López, C., Catalá, R., Turecková, V.,Novak, O., Zhang, W., Sieburth, L., Jiménez-Gómez, J.M., andSalinas, J. (2016). The LSM1-7 complex differentially regulatesArabidopsis tolerance to abiotic stress conditions by promotingselective mRNA decapping. Plant Cell 28: 505–520.

Pilet, P.E. (1975). Abscisic acid as a root growth inhibitor: Physio-logical analyses. Planta 122: 299–302.

Puranik, S., Sahu, P.P., Srivastava, P.S., and Prasad, M. (2012).NAC proteins: Regulation and role in stress tolerance. Trends PlantSci. 17: 369–381.

Qi, X., et al. (2014). Identification of a novel salt tolerance gene in wildsoybean by whole-genome sequencing. Nat. Commun. 5: 4340.

Qi, J., Song, C.P., Wang, B., Zhou, J., Kangasjärvi, J., Zhu, J.K.,and Gong, Z. (2018). Reactive oxygen species signaling and sto-matal movement in plant responses to drought stress and pathogenattack. J. Integr. Plant Biol. 60: 805–826.

Qin, C., Cheng, L., Shen, J., Zhang, Y., Cao, H., Lu, D., and Shen, C.(2016). Genome-wide identification and expression analysis of the14-3-3 family genes in Medicago truncatula. Front. Plant Sci. 7: 320.

Quach, T.N., Tran, L.S., Valliyodan, B., Nguyen, H.T., Kumar, R.,Neelakandan, A.K., Guttikonda, S.K., Sharp, R.E., and Nguyen,H.T. (2014). Functional analysis of water stress-responsive soybeanGmNAC003 and GmNAC004 transcription factors in lateral rootdevelopment in Arabidopsis. PLoS One 9: e84886.

Riechmann, J.L., et al. (2000). Arabidopsis transcription factors:Genome-wide comparative analysis among eukaryotes. Science290: 2105–2110.

Ruggiero, B., Koiwa, H., Manabe, Y., Quist, T.M., Inan, G.,Saccardo, F., Joly, R.J., Hasegawa, P.M., Bressan, R.A., andMaggio, A. (2004). Uncoupling the effects of abscisic acid on plantgrowth and water relations. Analysis of sto1/nced3, an abscisic

acid-deficient but salt stress-tolerant mutant in Arabidopsis. PlantPhysiol. 136: 3134–3147.

Sakuraba, Y., Kim, Y.S., Han, S.H., Lee, B.D., and Paek, N.C. (2015).The Arabidopsis transcription factor NAC016 promotes droughtstress responses by repressing AREB1 transcription through a tri-furcate feed-forward regulatory loop involving NAP. Plant Cell 27:1771–1787.

Saldanha, A.J. (2004). Java Treeview--extensible visualization of mi-croarray data. Bioinformatics 20: 3246–3248.

Sato, H., Takasaki, H., Takahashi, F., Suzuki, T., Iuchi, S., Mitsuda,N., Ohme-Takagi, M., Ikeda, M., Seo, M., Yamaguchi-Shinozaki,K., and Shinozaki, K. (2018). Arabidopsis thaliana NGATHA1 tran-scription factor induces ABA biosynthesis by activating NCED3gene during dehydration stress. Proc. Natl. Acad. Sci. USA 115:E11178–E11187.

Shahid, S.A., Zaman, M., and Heng, L. (2018). Soil salinity: Historicalperspectives and a world overview of the problem. In Guideline forSalinity Assessment, Mitigation and Adaptation Using Nuclear andRelated Techniques., M. Zaman, S.A. Shahid, and and L. Heng, eds(Cham, Switzerland: Springer), pp. 43–53.

Sharp, R.E., Poroyko, V., Hejlek, L.G., Spollen, W.G., Springer,G.K., Bohnert, H.J., and Nguyen, H.T. (2004). Root growth main-tenance during water deficits: Physiology to functional genomics.J. Exp. Bot. 55: 2343–2351.

Shi, W.Y., Du, Y.T., Ma, J., Min, D.H., Jin, L.G., Chen, J., Chen, M.,Zhou, Y.B., Ma, Y.Z., Xu, Z.S., and Zhang, X.H. (2018). The WRKYtranscription factor GmWRKY12 confers drought and salt tolerancein soybean. Int. J. Mol. Sci. 19: E4087.

Shu, Y.J., Song, L.L., Zhang, J., Liu, Y., and Guo, C.H. (2015).Genome-wide identification and characterization of the Dof genefamily in Medicago truncatula. Genet. Mol. Res. 14: 10645–10657.

Sirichandra, C., Gu, D., Hu, H.C., Davanture, M., Lee, S., Djaoui, M.,Valot, B., Zivy, M., Leung, J., Merlot, S., and Kwak, J.M. (2009).Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase byOST1 protein kinase. FEBS Lett. 583: 2982–2986.

Song, H., Yin, Z., Chao, M., Ning, L., Zhang, D., and Yu, D. (2014).Functional properties and expression quantitative trait loci forphosphate transporter GmPT1 in soybean. Plant Cell Environ. 37:462–472.

Song, Y., Ji, D., Li, S., Wang, P., Li, Q., and Xiang, F. (2012). Thedynamic changes of DNA methylation and histone modifications ofsalt responsive transcription factor genes in soybean. PLoS One 7:e41274.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S.(2013). MEGA6: Molecular evolutionary genetics analysis version6.0. Mol. Biol. Evol. 30: 2725–2729.

Tan, B.C., Joseph, L.M., Deng, W.T., Liu, L., Li, Q.B., Cline, K., andMcCarty, D.R. (2003). Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family. Plant J. 35: 44–56.

Tarazona, S., García-Alcalde, F., Dopazo, J., Ferrer, A., andConesa, A. (2011). Differential expression in RNA-seq: a matter ofdepth. Genome Res. 21: 2213–2223.

Tran, L.S., Nakashima, K., Sakuma, Y., Simpson, S.D., Fujita, Y.,Maruyama, K., Fujita, M., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2004). Isolation and functional analysis of Arabidopsisstress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1promoter. Plant Cell 16: 2481–2498.

Umezawa, T., Sugiyama, N., Takahashi, F., Anderson, J.C.,Ishihama, Y., Peck, S.C., and Shinozaki, K. (2013). Genetics andphosphoproteomics reveal a protein phosphorylation network in theabscisic acid signaling pathway in Arabidopsis thaliana. Sci. Signal.6: rs8.

GmSIN1 in Root Growth and Salt Tolerance 2129

Verslues, P.E., Agarwal, M., Katiyar-Agarwal, S., Zhu, J., and Zhu,J.K. (2006). Methods and concepts in quantifying resistance todrought, salt and freezing, abiotic stresses that affect plant waterstatus. Plant J. 45: 523–539.

Wang, F., et al. (2015a). GmWRKY27 interacts with GmMYB174 toreduce expression of GmNAC29 for stress tolerance in soybeanplants. Plant J. 83: 224–236.

Wang, J., et al. (2018). A single transcription factor promotes bothyield and immunity in rice. Science 361: 1026–1028.

Wang, P., and Song, C.P. (2008). Guard-cell signalling for hydrogenperoxide and abscisic acid. New Phytol. 178: 703–718.

Wang, N., Zhao, S.Z., Lv, M.H., Xiang, F.N., and Li, S. (2016). Re-search progress on identification of QTLs and functional genes in-volved in salt tolerance in soybean. Yi Chuan 38: 992–1003.

Wang, T., Tohge, T., Ivakov, A., Mueller-Roeber, B., Fernie, A.R.,Mutwil, M., Schippers, J.H., and Persson, S. (2015b). Salt-relatedMYB1 coordinates abscisic acid biosynthesis and signaling duringsalt stress in Arabidopsis. Plant Physiol. 169: 1027–1041.

Wei, W., Huang, J., Hao, Y.J., Zou, H.F., Wang, H.W., Zhao, J.Y.,Liu, X.Y., Zhang, W.K., Ma, B., Zhang, J.S., and Chen, S.Y. (2009).Soybean GmPHD-type transcription regulators improve stress tol-erance in transgenic Arabidopsis plants. PLoS One 4: e7209.

Wu, A., et al. (2012). JUNGBRUNNEN1, a reactive oxygen species-responsive NAC transcription factor, regulates longevity in Arabi-dopsis. Plant Cell 24: 482–506.

Wu, X., Gaffney, B., Hunt, A.G., and Li, Q.Q. (2014). Genome-widedetermination of poly(A) sites in Medicago truncatula: Evolutionaryconservation of alternative poly(A) site choice. BMC Genomics 15:615.

Xie, Y.J., Xu, S., Han, B., Wu, M.Z., Yuan, X.X., Han, Y., Gu, Q., Xu,D.K., Yang, Q., and Shen, W.B. (2011). Evidence of Arabidopsissalt acclimation induced by up-regulation of HY1 and the regulatoryrole of RbohD-derived reactive oxygen species synthesis. Plant J.66: 280–292.

Xu, G., Yuan, M., Ai, C., Liu, L., Zhuang, E., Karapetyan, S., Wang,S., and Dong, X. (2017). uORF-mediated translation allows en-gineered plant disease resistance without fitness costs. Nature 545:491–494.

Yabuta, Y., Morishita, T., Kojima, Y., Maruta, T., Nishizawa-Yokoi,A., and Shigeoka, S. (2010). Identification of recognition sequenceof ANAC078 protein by the cyclic amplification and selection oftargets technique. Plant Signal. Behav. 5: 695–697.

Yang, Y., and Guo, Y. (2018). Unraveling salt stress signaling inplants. J. Integr. Plant Biol. 60: 796–804.

Yang, L., Zhang, J., He, J., Qin, Y., Hua, D., Duan, Y., Chen, Z., andGong, Z. (2014). ABA-mediated ROS in mitochondria regulate rootmeristem activity by controlling PLETHORA expression in Arabi-dopsis. PLoS Genet. 10: e1004791.

Yang, S.D., Seo, P.J., Yoon, H.K., and Park, C.M. (2011). The Ara-bidopsis NAC transcription factor VNI2 integrates abscisic acidsignals into leaf senescence via the COR/RD genes. Plant Cell 23:2155–2168.

Yang, X., Wang, X., Ji, L., Yi, Z., Fu, C., Ran, J., Hu, R., and Zhou, G.(2015a). Overexpression of a Miscanthus lutarioriparius NAC geneMlNAC5 confers enhanced drought and cold tolerance in Arabi-dopsis. Plant Cell Rep. 34: 943–958.

Yang, Y., Sun, T., Xu, L., Pi, E., Wang, S., Wang, H., and Shen, C.(2015b). Genome-wide identification of CAMTA gene family mem-bers in Medicago truncatula and their expression during root nodulesymbiosis and hormone treatments. Front. Plant Sci. 6: 459.

Yu, Y., Liu, Z., Wang, L., Kim, S.G., Seo, P.J., Qiao, M., Wang, N., Li,S., Cao, X., Park, C.M., and Xiang, F. (2016). WRKY71 acceleratesflowering via the direct activation of FLOWERING LOCUS T andLEAFY in Arabidopsis thaliana. Plant J. 85: 96–106.

Zhai, Y., Wang, Y., Li, Y., Lei, T., Yan, F., Su, L., Li, X., Zhao, Y., Sun,X., Li, J., and Wang, Q. (2013). Isolation and molecular character-ization of GmERF7, a soybean ethylene-response factor that in-creases salt stress tolerance in tobacco. Gene 513: 174–183.

Zhang, D.P. (2014). Abscisic Acid: Metabolism, Transport and Sig-naling. (Dordrecht, The Netherlands: Springer).

Zhang, W., et al. (2019). A cation diffusion facilitator, GmCDF1,negatively regulates salt tolerance in soybean. PLoS Genet. 15:e1007798.

Zhang, K., and Gan, S.S. (2012). An abscisic acid-AtNAP transcrip-tion factor-SAG113 protein phosphatase 2C regulatory chain forcontrolling dehydration in senescing Arabidopsis leaves. PlantPhysiol. 158: 961–969.

Zhang, C., Bousquet, A., and Harris, J.M. (2014). Abscisic acidand lateral root organ defective/NUMEROUS INFECTIONS ANDPOLYPHENOLICS modulate root elongation via reactive oxygenspecies in Medicago truncatula. Plant Physiol. 166: 644–658.

Zhang, G., Chen, M., Li, L., Xu, Z., Chen, X., Guo, J., and Ma, Y.(2009). Overexpression of the soybean GmERF3 gene, an AP2/ERFtype transcription factor for increased tolerances to salt, drought,and diseases in transgenic tobacco. J. Exp. Bot. 60: 3781–3796.

Zhang, X., Zhang, L., Dong, F., Gao, J., Galbraith, D.W., and Song,C.P. (2001). Hydrogen peroxide is involved in abscisic acid-inducedstomatal closure in Vicia faba. Plant Physiol. 126: 1438–1448.

2130 The Plant Cell

DOI 10.1105/tpc.18.00662; originally published online June 21, 2019; 2019;31;2107-2130Plant Cell

Xiao, Ming-yi Bai and Fengning XiangLangtaoLyu, Juanjuan You, Yangyang Zhang, Luli Wang, Xiaofang Wang, Zhenhua Liu, Jianhua Tong,

Shuo Li, Nan Wang, Dandan Ji, Wenxiao Zhang, Ying Wang, Yanchong Yu, Shizhen Zhao, MenghuaRegulates Root Growth in Soybean Exposed to Salt Stress

A GmSIN1/GmNCED3s/GmRbohBs Feed-Forward Loop Acts as a Signal Amplifier That

 This information is current as of January 7, 2020

 

Supplemental Data /content/suppl/2019/06/21/tpc.18.00662.DC1.html /content/suppl/2019/07/17/tpc.18.00662.DC2.html

References /content/31/9/2107.full.html#ref-list-1

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