Molecular Networking of Regulated Transcription Factors Under Salt Stress in Wild Barley (H. spontaneum)

Salt stress is among environmental conditions that severely retards plant growth. Scope of this work is the detection of transcription factors that might participate in regulating salt-stressed genesin wild barley (Hordeumspontaneum). Expression profiles of important types of transcription factors (TFs) were displayed. They includeWRKY and MYB, that were regulated under salt stress. WRKY19 and NAC96 are known to induce stress tolerance through activation of DREB2A (or Ap2-ERF). NAC96 concordantly upregulated with DREB2A gene under salt stress in H. spontaneum, a possible cross talking to compensate the negative performance of WRKY19 gene. P5CS, for proline accumulation, is also known to be driven by ERF1 and genes encoding these proteins concordantly upregulated in H. spontaneum under salt stress supporting NAC96/ERF1/P5CS cross talking towards proline accumulation under stress. Genes encoding enzymes participating in the last steps of glucose, sucrose and maltose biosyntheses concordantly upregulated with WRKY11 that is also involved in driving genes encoding free proline. B-box zinc finger protein 21 (BZF21) concordantly expressed with genes encoding catalase and SAUR40 indicating that BZF21 gene might drive expression of the two genes under salt stress. Upregulated WRKY41 and WRKY46 under salt stress in wild barley are known to exhibit enhanced stomatal closure, reactive oxygen species (ROS) scavenging, lateral roots development via regulation of ABA signaling and auxin homeostasis. The latter action is governed by GH3.8 gene that was upregulated in wild barley. MYB30 is known for being SUMOylated by SIZ1. In the present study, MYB30, MYB44 and MYB3R-2 genes were concordantly expressed with SIZ2 gene supporting their crosstalking under salt stress in H. spontaneum. Based on the regulation of WRKY19 and MYB30 genes under salt stress in H. spontaneum, we suggest that the first is a positive activator, while the second is a negative activator of FT gene that drives early flowing in plants. MYB44 that promotes stomatal closure under stress can also serve in conferring tolerance to abiotic stresses in wild barley. Several other downregulated genes under salt stress, e.g., MYB1, MYB20 and MYB73, were previously reported to negatively regulate abiotic stress tolerance in plants. We suggest that WRKY gene family participates in salt stress responses in leaves of H. spontaneum following approaches different from those of other plants. Regulation of MYB gene family is almost similar to that of other plant species under salt stress. In conclusion, the present study addresses some of the regulatory frame works driving expression of salt-related genes inH. spontaneum that can be utilized in plant, e,g, cereals, breeding programsto improve their salt stress tolerance.

expression levels of different stress-related genes are regulated by transcription factors (TFs) that work as stimulators of individual genes or act as master switches driving a battery of genes or a whole pathway such as stress signal transduction pathways [2][3][4][5][6][7][8] WRKYis among the largest families of TFs that play important roles in modulating physiological processes in plants under stress conditions [8][9][10][11][12] . Protein encoded by this TFgene family is characterized by a 60 amino acids domain of highly conserved WRKYGQKheptapeptide at the N-terminal and an atypical zinc finger-like motif at its C-terminal 13,14 . This family contains over 70 members in Arabidopsis 13,15 . 55 in cucumber 16 , 119 in maize 17 , 94 in barley 18 , and 100 in rice 19 . Encoded proteins of this family are characterized by a 60-amino acids domain containing the WRKY amino acid sequence at its amino-terminal end and a putative zinc finger motif at its carboxy-terminal end. Based on number and diversity of WRKY domains, WRKY proteins are classified into three groups (I, II and III) of which category I proteins harbor two domains, while proteins of groups II and III harboronly one domain. Groups II and III proteins differ in zinc finger structures (C2H2 in group II, while C2HC in group III) 14,20 . Previous study reported a number of 74 WRKY proteins in Arabidopsis, while over 100 in rice (Oryza sativa) 10 . WRKYTFs have specificity to bind W-box [TTGAC(C/T)] of promoters of their target genes, which subsequently wire genetic circuits towards downstream biological responses 20,21 .
WRKYTFs can either negatively or positively trigger a certain response under stress conditions 19 . These regulation patterns as well as members participating in a given condition can changes from a plant to the other. For example, WRKY54 and WRKY70 in Arabidopsis negatively regulate leaf senescence 22 . While, WRKY23 positively enhanced pathogen defense and over expression of maize WRKY58 in rice 23 . and wheat WRKY1 and WRKY33 in Arabidopsis positively conferred drought and salt tolerance 24 . WRKYTFs also reported to be involved in abiotic stress by wiring ABA signaling pathway 25 . For example, Chrysanthemum WRKY1 enhancedabiotic stress tolerance, whilecotton WRKY17 overexpressed in tobacco reduced tolerance by regulating a number of genes in ABA signaling pathway and reactive oxygen species (ROS) production.
MYB is also a family of TFs involved in response to abiotic stresses in plants 26 . Of which, expression of MYB108 gene in Arabidopsis is induced in response to salt stress and participate in crosstalking between abiotic and biotic stresses via orchestration of signaling pathways of jasmonic acid (JA) and gibberellic acid (GA) 27,28 . While, MYB65 participates in GA signaling in growth and flowering processes 29 . Our work also showed that the expression of this gene increased in roots in response to both stresses whereas, in leaves upregulated only in response to salt stress. Expression of genes encoding MYB differs in different tissues in response to salt stress as MYB34 in Arabidopsis, for example, is normally upregulated in root tissue and its expression in leaves increases only in response to stress, whereas MYB47 and MYB32 were common in both tissues 28,30 .
In the present study, we have demonstrated important types of TFs including WRKY and MYB that were regulated under salt stress in wild barley Hordeumspontaneum. The information recovered from this work can be helpful in improving plant salt stress tolerance in the future.

Materials and Methods
Salt stress experiment was conducted on H. spontaneum as previously described 31 . Fourteen-day-old seedlings were treated with salt (500mMNaCl) and total RNAs were harvested in a replicated experiment from leaves at 0 (control), 2, 12 and 24 h time point using Trizol (Invitrogen, Life Tech, Grand Island, NY, USA). Then, RNAs were treated with RNase-free DNase (Promega Corporation, Madison, WI, USA) and 1 U/ul of RNasin® Plus RNase Inhibitor as described (Promega Corporation, Madison, WI, USA). Total RNA samples were, then, shipped to Beijing Genomics Institute (BGI), Shenzhen, China for deep sequencing using illuminaMiseq. Generated raw data were retrieved in FASTQ format and submitted to the NCBI and experiment received accession number of PRJNA227211 (https://www. ncbi.nlm.nih.gov/ bioproject?LinkName=sra_ bioproject&from_uid=537429). Individual accession numbers of raw data of different samples are available in NCBI (https://www.ncbi. nlm.nih.gov/ sra?LinkName=bioproject_sra_ all&from_uid=227211). Raw data was processed as described 32 and clean data was subjected to genomeguided Trinity de novo transcriptome assembly (https://github.com/trinityrnaseq/trinityrnaseq/ wiki/Genome-Guided-Trinity-Transcriptome-Assembly) with Hordeumvulgare genome (https:// plants.ensembl.org/ Hordeum_vulgare/Info/ Index, Taxonomy ID 112509) used as the guide. Differential expression and cluster analysis were done by EdgeR (version 3.0.0, R version 2.1.5) with proper algorism and fold change values of e" 2 measured against actin house-keeping gene. Annotation of the recovered transcripts was done using Blast2GO (http://www.blast2go.org/). Subsequent bioinformatics approach was done as described 33 . Predicted CDSs were annotated against protein database in order to assign functions of transcripts. Protein domains common in TFswere identified using HMMER3 software 34 .
Then, RNA-Seq datasets were validated via qRT-PCR of four randomly selected genes using the Agilent Mx3000P qPCR Systems (Agilent technology, USA) as previously described 31 . Transcripts selected from cluster analysis were upregulated at 2 and 12 h time points. Primer sequences are shown in Table S1. Calculations referring to expression levels of each transcript were done relative to that under control condition and barley actin gene was used as the housekeeping gene.

results and discussion
For validating RNA-Seq datasets, qRT-PCR was done for four randomly selected transcripts encoding transcripts that were either upregulated at 2 and 12 h time points, upregulated at 2 h time point, or downregulated at 2 and 12 h time points of salt stress and results aligned with RNA-Seq datasets for transcripts used for validation (FigureS1).Cluster analysis resulted in the recovery of over 10000 differentially expressed (DE) transcripts with fold change of e" 2 under salt stress including over 600 TFs highlighted in Table  S2that are separately shown in Table S3. Wellknown TF families for their response to abiotic stresses include WRKY and MYB. Genes encoding WRKY activated at 2 and 12 h time points under salt stress include WRKY2, WRKY11, WRKY41, WRKY46, WRKY50 and WRKY71 (Figure 1a). Gene encoding WRKY24 was upregulated at 2 h  (Table S3), STZ gene was not regulated. Then, STZ gene cannot be used in tracing regulation of WRKY2 gene.
WRKY19gene was downregulated in leaves of H. spontaneumunder salt stress (Figure 1b), thus, no activation of DREB2Agene is expected. DREB2A is among AP2-ERF (Apetala2/Ethylene responsive factor) gene family and a recent report indicated that DREB2A is also affected by other TFs, ex., NAC96 (28). Interestingly, two Ap2-ERFgene isoforms and a gene encoding NAC96 were upregulated in cluster 1 under salt stress in leaves of H. spontaneum( Figure 3 and Table S2) indicating that upregulation of Ap2-ERF gene can compensate the negative regulation of WRKY19gene in H. spontaneum.
Overexpression of WRKY2gene in grapevine increased proline under salt stress 36 . Two other recent reports indicated that WRKY2 in wheat 37 and WRKY11 in soybean 38 also drive genes encoding free proline and soluble sugars  Table S2 under drought stress. Prolineand sugars are known as important osmolytesthat neutralize the effects of salt and alleviate stress 39,40 . Interestingly, gene encoding Delta-1-pyrroline-5-carboxylate synthase (P5CS) for proline accumulation was concordantly upregulated under salt stress with that encoding ERF1 (ethylene responsive factor 1)gene, otherDREBgene derivative, in cluster 32 ( Figure  4 and Table S2). Then, proline accumulation due to the function of P5CSgene in H. spontaneum under salt stress canalso be driven by ERF1gene that is likely controlled by NAC96, not by either WRKY2 or WRKY11. As per expected sugar levels under salt stressin H. spontaneum, results indicated that genes encoding enzymes participating in the last step of glucose (e.g., beta-glucosidase), sucrose (e.g., sucrose synthase 6) and maltose (e.g., beta-amylase 8) biosynthesis concordantly upregulated with WRKY2 in clusters1 and 5, whileWRKY11 and MYB3R-2 in cluster 6 ( Table  S2) under salt stress ( Figure 5).Accordingly, we speculate that WRKY 2 and WRKY11 are involved  Table S2.  Table S2 in driving genes encodingsoluble sugars as two different mechanisms of salt stress tolerance in H. spontaneum.
Recently, WRKY11 was also proven to induce elevated levels of superoxide dismutase (SOD) and catalase in soybean 38 . In the present study, upregulated WRKY11gene does not seem to concordantly express with SOD regulatedgene isoforms in H. spontaneum, whereSOD gene isoforms were downregulated ( Figure 6 and Table  S2) as shown in clusters 2 and 27 and no other TFcan likely complementWRKY11 effect whose upregulation pattern of its three isoforms inH.
spontaneum was different (cluster 1). Although both genes are upregulated, WRKY11gene does not either concordantly express with isoforms of gene encoding catalase(existing in cluster 23), but gene encoding another TF namely B-box zinc finger protein 21 (BZF21) concordantly expressed with the two isoforms of gene encoding catalase, thus, possibly drive expression of this gene in H. spontaneuminstead of WRKY11 (Figure 7 and Table S2). B-box zinc finger proteins were reported to enhance salt and drought stresses tolerance in Arabidopsis (Liu et al., 2019). Interestingly, gene encoding SAUR40also concordantly expressedwith  Table S2 BZF21 and catalase genes in cluster 23 ( Figure 7 and Table S2).SAUR40gene is among a family acting as a regulator of cell elongation and plant growth performance 41 and a stimulator of shoot elongation due to auxin signaling 42 . Thus, we speculate that BZF21 might drive expression of both catalase and SAUR40 genes as genes encoding the three metabolites are concordantly expressed (Figure 7). 43 . However, expression patterns of isoforms of these two TFs seem to be controversial (Figure 1) as gene encoding the first was upregulated at 2 h time point and downregulated at 24 h time point, while gene encoding the second was upregulated at 2 and 12 time points. Xie et al 19 Table S2 WRKY24gene is induced by ABA signaling, while Basu and Roychoudhury 43 indicated that ABA signaling induces higher expression of WRKY71gene and many other TFs. Interestingly, the authors indicated thatWRKY24gene showed expression even lower than that of the control untreated samples under salt stress. WRKY71 was recently reported to antagonistically act against both salt-delayed flowering and escaping salt stress in Arabidopsis through the induction of gene encoding FLOWERING LOCUS T (FT) 44 .

indicated that
Surprisingly, two isoforms of the latter gene in clusters 4 and 8 seem concordantly downregulated with gene encoding WRKY19of cluster 4 rather than with gene encoding WRKY71of cluster 21 ( Figure 8 and Table S2). We cannot jump to conclusions on the relationship between WRKY19 and FT genes unless an experiment to detect the consequences of WRKY19 gene being knocked out in Arabidopsis model.
Aligning with the results of the present study, WRKY41and WRKY46 were reported to  Table S2 positively relateto salt stress tolerance in tobacco 45 . Genes encoding the two TFs were upregulated under salt stress in H. spontaneum as shown in clusters 11 and 1, respectively (Figure 1 and Table  S2). Overexpressing the cotton WRKY41gene in tobacco exhibited enhanced stomatal closure and reactive oxygen species (ROS) scavenging when plants were exposed to osmotic stress 45 . WRKY46acts in Arabidopsis in developinglateral roots under osmotic/salt stress via regulation of ABA signaling and auxin homeostasis 46 . Auxin homeostasis is known to be regulated by GRETCHEN HAGEN3 or GH3 gene family in "Plant hormone signal transduction" pathway 42 . In the present study, GH3.8gene was upregulated in cluster 17 with no exact TF concordantly expressed with it ( Figure 9 and Table S2). No conclusive information on the function of WRKY50 (cluster 1) is available except that it acts as a positive regulator in the salicylic acid (SA) signaling pathway and  Table S2 probably ABA signaling pathway in Arabidopsis, while a negative regulator in jasmonic acid (JA) signaling 47,48 .Very little is known about the mode of action of WRKY35 except that its expression participates in conferring salt stress tolerance in zoysia grass 49 .We conclude that WRKY gene family participates in salt stress responses in leaves of H. spontaneum in ways different from those in other plant species.
Expression of five MYB genes,namely MYB30, MYB44, MYB62, MYB3R-2 and MYB3R-4, was proven to be increased under salt stress in leaves of H. spontaneum (Figure 2a), while expression of four, namely MYB1, MYB20, MYB73 and MYBS3, wasdecreased (Figure 2b). MYB30, anR2R3 MYBTF, was studied by Gong et al 50 and results indicated that expression in the perennial wall-rocket (Diplotaxistenuifolia L.) increased under salt stress up to 4 h time point, while gradually decreased up to 24 h time point in perfect alignment with results of the present study with regard to regulation of gene encoding this TF. MYB30 was proven to be SUMOylated by SIZ1 (50,51). SUMOylation represents a post-translational regulation involved in various cellular processes including response to stresses 52 . SmallUbiquitin-likeModifier (SUMO) proteins, like SIZ1 and SIZ2,represent a family of smallproteinscovalentlyattached to a certain protein, while detached from others to modify target protein's(e.g., MYB30) function. In the present study, two isoforms of MYB30gene as well as MYB44 and MYB3R-2 genes concordantly expressed with SIZ2gene in cluster 6 under salt stress in H. spontaneum (Figure 10and Table S2). MYB30also accelerates flowering both in long and short days. Early flowering is mediated by elevated expression ofFLOWERING LOCUS T(FT)gene that is mainly activated byCONSTANS (CO). However, MYB30can also drive expression of FTgene 53 , a phenomenon that we speculated for WRKY19gene under salt stress in H. spontaneum. The major difference between the possible regulation of WRKY19 orMYB30gene is that the first is a positive activator, while the second is a negative activator of FT gene. This controversial speculated regulation ofWRKY19 and MYB30genes under salt stress in H. spontaneum is shown in Figure11.MYB30was alsoreported to participate in ABA signaling response 51 , in accumulation of very-long-chain fatty acids such as waxes, phospholipids, and complex sphingolipids 54 , and in promoting the expression of a subset of brassinosteroids (BRs) target genes 55,56 . No results were detected on the regulation of genes encoding any of the above-mentioned compounds under salt stress in H. spontaneum.
Interestingly, MYB44 was proven to be a negative regulator of ABA signaling and abiotic stresses in Arabidopsis 57 , while positively increased sensitivity of seed germination to ABA 58 . The latter authors indicated that phosphorylation of MYB44 by MAPK is mandatory for its function. Nonetheless, Jung et al 59 indicated that MYB44 promotes stomatal closure, a characteristic shared with WRKY41 that consequently serves in conferring tolerance to abiotic stresses in Arabidopsis.Tolerance is conferred for plants overexpressing MYB44 gene because they exhibit a reduced rate of water loss, reduced rate of genes encoding serine/threonine protein phosphatases 2C (PP2Cs), then enhanced tolerance to drought and salt stress. Nonetheless, genes encoding MYB44, protein phosphatase 2C and two isoforms of protein phosphatase 1 (PP1)in the present study are concordantly expressed in cluster 6 ( Figure 12 and Table S2). Explanation of concordant expression of MYB44 and genes encoding the two phosphatases might be that MYB44 was upregulated only at 2 h time point only, while the other genes were upregulated also at 12 h time point. This indicates that negative regulation of MYB44 might take place only at 12 h time point.
Devaiah et al 60 stated that MYB62is actingtowards suppression of several phosphate (Pi) starvation-induced genes and suppression of gibberellic acid (GA) biosynthesis under nutrient stress. Authors claimed that cross-talking between Pi homeostasis and GA is an adaptive mechanism underabiotic stresses. Therefore, it is logic that MYB62 negatively regulate expression of gibberellin-regulated protein under salt stress in H. spontaneum as previously described 60 . Inthe present study, MYB62 exists in cluster 1 whose expression of transcripts indicated upregulation at 2 and 12 h time points, while one gibberellinregulated protein exists in cluster 4 whose expression of transcripts indicated downregulation at 2 and 12 h time points (Figure 13 and Table  S2). As per MYB3R-4, Haga et al 61 indicated its participation in pleiotropic development and regulation of multiple G2/M-specific genes in Arabidopsis. None of the genes involved in the latter processes were regulated in H. spontaneum under salt stress.
MYB1, MYB20, MYB73 and MYBS3genes were shown to be downregulated under salt stress in H. spontaneum (Figure 2b). These TFs were previously reported to negatively regulate abiotic stress tolerance in plants except forMYBS3 that was reported for its positive role in abiotic stresses, particularly cold stress tolerance in rice via mediation of a-amylase gene expression 62 .
In H. spontaneum, MYBS3 seems concordantly expressed with a-amylase gene although the first exists in cluster 4, while the second exists in cluster 2 ( Figure 14 and Table S2). Wang et al 63 claimed that MYB1 negatively regulates seed germination under saline conditions in Arabidopsis by regulating the levels of the stress hormone abscisic acid (ABA). Similar conclusions were reached by Gao et al 47 in their work on MYB20 in Arabidopsis with regard to the negative regulation of ABA under drought stress. Loss-of-function experiment of MYB73gene resulted in drought or/ and salt tolerance due to its negative regulation of SOS1 (salt overly sensitive 1) and SOS3 genes (64,65). No SOS genes are regulated under salt stress in H. spontaneum.
Summary of the overall molecular networking involving transcription factors and their concordantly expressed genes along with downstream biological processes towards conferring salt stress tolerance in H. spontaneum under salt stress is shown in Figure 15.

conclusion
In conclusion, we suggest that WRKY gene family participates in salt stress responses in leaves of H. spontaneum of which some of them follow different approaches, in terms of their regulation under salt stress as well as the downstream responsive genes, from those of other plant species. Regulation of MYB gene family in H. spontaneumseems similar, to a large extent, to that of other plant species under salt stress. The present study addressed some of the molecular mechanisms by which H. spontaneum follows under salt stress in order to stand severe salt stress. One of the important avenue towards improving salt stress tolerance is understanding the regulatory elements, e.g. transcription factors, that drive important saltrelated genes. This information might be useful in subsequent breeding programs in cultivated barley and other cereal crops.