Heliyon 5 (2019) e02614

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Heliyon
journal homepage: www.heliyon.com

Research Article

Microarray analysis of transcriptional responses to salt and drought stress in

Arabidopsis thaliana
Razieh Ghorbani, Abbas Alemzadeh *, Hooman Razi
Department of Crop Production and Plant Breeding, School of Agriculture, Shiraz University, Shiraz, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Microarray expression profile analysis is a useful approach to increase our knowledge about genes involved in
Agriculture regulatory networks and signal transduction pathways related to abiotic stress tolerance. Salt and drought, as two
Plant biology important abiotic stresses, adversely affect plant productivity in the world every year. To understand stress
Ecology
response mechanisms and identify genes and proteins which play critical roles in these mechanisms, the study of
Genetics
Agronomy
individual genes and proteins cannot be considered as an effective approach. On the other hand, the availability of
Horticulture new global data provides us an effective way to shed some light on the central role of molecules involved in stress
Lagenaria siceraria response mechanisms in the plant. A meta-analysis of salt and drought stress responses was carried out using 38
Cucurbits samples of different experiments from leaves and roots of Arabidopsis plants exposed to drought and salt stresses.
Harvest time We figured out the number of differentially expressed genes (DEGs) was higher in roots under both stresses. Also,
Vivipary we found that the number of common DEGs under both stresses was more in roots and also the number of
Seed maturation common DEGs in both tissues under salt stress was more than drought stress. The highest percent of DEGs was
Yield
related to cell and cell part (about 87%). Around 9% and 7% of DEGs in roots and leaves encoded transcription
Arabidopsis
factors, respectively. Network analysis revealed that three transcription factor families HSF, AP2/ERF and C2H2,
Meta-analysis
Transcriptomics may have critical roles in salt and drought stress response mechanisms in Arabidopsis and some proteins like STZ
Transcription factors may be introduced as a new candidate gene for enhancing salt and drought tolerance in crop plants.

1. Introduction different stresses [4]. Molecular biology techniques together with bio-
informatic studies have provided a powerful tool to identify a number of
Plants unlike animals are not able to move and escape from unde- genes and proteins involving in different pathways of stress response
sirable condition and stresses. Abiotic stresses such as drought, salt, mechanisms (SRMs) [5, 6]. Molecular response of plants to environ-
extreme temperature, heavy metals and nutrient deficiency have unde- mental stresses may actually involve a complex interaction between
sirable effects on plants [1]. These stresses cause decrease in growth and different molecules related to various pathways. Meta-analysis is the
productivity of plants. Among these stresses, drought and salt are two statistical approach which combine results from different studies to
major environmental factors which have detrimental effects on growth, clarify simultaneously expression of thousands of genes and could be
yield and crop production. There is severe water shortage in the world used to detect key genes in response to different stresses [7]. Recently,
and the majority of these water resources are exposed to salt. Hence, it is meta-analysis methods enable scientists to simultaneously study different
necessary to improve water use efficiency and salt tolerance of crops molecules such as transcripts and proteins under various conditions [8].
through plant breeding projects using new tools like biotechnology [2]. The stress-inducible genes that their products involved in the stress
Plant have responded to salt and drought stresses at the molecular may be classified into two groups: the first group including those that
and cellular levels and also at the physiological and biochemical levels protect the cells against stress; and the second group including those that
[3]. There are different mechanisms evolved in plants to receive and have crucial roles in gene expression under stress conditions [9].
transmit signals from the environment to cells which leads to response to Determining common proteins and their encoding genes between

* Corresponding author.
E-mail address: [email protected] (A. Alemzadeh).

https://doi.org/10.1016/j.heliyon.2019.e02614
Received 10 April 2019; Received in revised form 24 July 2019; Accepted 4 October 2019
2405-8440/© 2019 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
R. Ghorbani et al. Heliyon 5 (2019) e02614

different stresses will allow us to shed some light on the SRMs in plants. 2.2. Detection of DEGs
Recent years have witnessed an upsurge in the number of functional
molecular studies reporting crosstalk in different pathways involved in The data were retrieved from ArrayExpress database and then
stress response mechanisms. There are some meta-analysis data from analyzed to obtain DEGs involved in responses to drought and salt
different plants revealing important genes which have hub roles in stresses. The downloaded raw data in format CEL data were analyzed by
different abiotic stresses [6, 10]. Among the plants, Arabidopsis as a FlexArray software version 1.6.3. Data were normalized by Robust Multi-
model plant has been subjected to a number of meta-analysis studies array Average (RMA) algorithm using R version 3.2.2. To identify DEGs,
which leads to finding differentially expressed genes (DEGs) between the processed microarray data were subjected to two-sample student's t-
stress and non-stress conditions [8, 11, 12, 13]. Detection of DEGs is the test. Up-regulated genes with significant P-value (false discovery rate
first step in understanding of SRMs in plants [13]. Recent advances in (FDR) < 0.05) with fold change value greater than 1.5 selected as DEGs
biotechnology and development of new technologies caused to facilitate [6, 11]. Venn diagram of DEGs was created by Venny 2.0 (http://bioinfo
the detection of new genes and determine their functions. Genomics, gp.cnb.csic.es/tools/venny/).
transcriptomics, proteomics and metabolomics may be used to identify
important genes related to abiotic stress response and determine their 2.3. Detection of transcription factors
roles in different pathways involved in SRMs [14]. One of the most
important molecules which play crucial roles in these mechanisms are The selected DEGs were further compared with the transcription
transcription factors [6]. There are some reports showed the roles of factors genes in the PlantTFDB server http://planttfdb.cbi.pku.edu.cn/i
some transcription factors family in regulation of stress-inducible genes ndex.php/ for identification of genes encoding transcription factors.
under stress conditions [6, 9, 15].
Gene ontology (GO) enrichment has been used as a powerful tool in 2.4. Gene ontology of DEGs
many studies to determine the molecular activity of a gene (molecular
function), determine the physiological role of a gene product and coor- GO enrichment analysis of DEGs was performed using the AgriGO
dination with other genes (biological process) and determine the location tool (http://bioinfo.cau.edu.cn/agriGO/) with default parameters such
of gene product in the cell (cellular component). Also, GO is usually used as Fisher statistical test method, Multi-test adjustment method of Yeku-
to annotate the function of genes and their proteins from various species tieli (FDR under dependency) with significant level of 0.05 and complete
especially model organisms such as Arabidopsis. GO gene ontology type [16]. A Singular Enrichment Analysis (SEA) was
The aim of this study was to identify genes involved in drought and performed using TAIR genome as reference and FDR less than <0.05.
salt stress responses in Arabidopsis using microarray gene expression data
by meta-analysis approach. In addition, we aimed to detect DEGs be- 2.5. Interaction networks between DEGs
tween stress and non-stress conditions. Moreover, in this research it was
tried to figure out most important transcription factors involved in theses Functional associations between DEGs during drought and salt
stresses that affect the expression of various genes through their down- or stresses were figured out by STRING v.10 (Search Tool for the Retrieval
up-regulation. In this paper, the functions of identified DEGs and tran- of Interacting Genes/Proteins) database (https://string-db.org/). These
scription factors were discussed and using network and GO analysis, the associations were constructed based on co-expression data which is
complexity of SRMs in Arabidopsis was explained. stored in the NCBI-GEO database, co-occurrence of the genes in the same
organisms, text mining which is a list of significant protein interaction
2. Materials and methods groups, extracted from the abstracts of scientific literature, significant
protein interaction datasets from other experiments, individual gene
2.1. Gene expression data collection fusion events per species, significant protein interaction groups gathered
from curated databases and neighborhood which is runs of genes that
The raw microarray expression data was downloaded from ArrayEx- occur repeatedly in close neighborhood in genomes. Confidence score for
press database (https://ebi.ac.uk/arrayexpress/) to identify drought and interactions was medium score (above 0.4). The gene network obtained
salt stress-responsive genes in Arabidopsis. The data originated from from STRING v.10 was imported into Cytoscape version 3.6.0 for further
Affymetrix microarray platform. These datasets obtained from 6 inde- analysis and display.
pendent experiments with 38 samples from leaves and roots of plants
exposed to drought and salt stresses. These data were obtained from 3. Results and discussion
series IDs E-MEXP-1863, E-GEOD-48474 and E-GEOD-40061 (response
to drought stress), and E-MEXP-2858, E-GEOD-53308 and E-GEOD- 3.1. Identification of DEGs in Arabidopsis thaliana under stress conditions
71001 (response to salt stress). All experiments were performed under
22–25
C, 65–90% RH (relative humidity) and 16/8 h light/dark cycle in This study was carried out to achieve more information about the
the growth chamber. Wild-type Columbia-0 was used in all experiments. changes in gene expression in response to drought and salt stresses in
In the E-MEXP-1863 experiment, 3-week-old plants were subjected to leaves and roots of Arabidopsis. Through comprehensive comparison
drought stress by withholding water for 5 days until plant samples analysis, we identified a set of 4540 up-regulated DEGs in the leaf tissue
reached of 52.5  7.5% relative water content (RWC). In the E-GEOD- which 1643 (36%) and 2897 (64%) of them up-regulated in response to
48474 experiment, drought stress was applied to 2-week-old WT plants drought and salt stress, respectively, whereas in the root tissue 6906
were by withholding water for 21 days. Leaf sample were collected for DEGs were up-regulated that 3150 (46%) and 3756 (54%) of them up-
transcriptome analysis in mentioned experiments. In the E-GEOD-40061 regulated in response to drought and salt stress, respectively (supple-
experiment, root sample were collected from 3-week-old plants which mentary 1). The number of DEGs which up-regulated in roots was 1.5-
were exposure under drought stress using withholding water for 14 days fold higher than leaves that under drought stress was 1.9-fold, whereas
(soil moisture below 30%). In the E-MEXP-2858 experiment, leaf sample under salt stress was less than 1.3-fold. A four-way Venn diagram was
were collected from one-month plants which treated with 200mM NaCl used to represent co-occurrence of DEGs under salt and drought stresses
for seven days for salt treatment. In the E-GEOD-53308 experiment, leaf in root and leaf tissues. The diagram represented a list of 584 (13%) DEGs
sample were collected from one-month plants which treated with 150 was up-regulated under salt and drought stresses in leaf tissue, but 1186
mM NaCl for four days for salt treatment. In the E-GEOD-71001 experi- (17%) DEGs were common between drought and salt stresses in roots
ment, root sample were collected from 6-week-old plants which treated (Fig. 1). In the other words, the number of DEGs involved in both stresses
with 150 mM NaCl for 6 h. in roots was 2-fold higher than leaves. Also, the common DEGs between

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roots and leaves under drought and salt stresses were determined using respectively in drought and salt stresses in leaves and 3026 and 3628
the Venn diagram. Under salt stress, 1266 (11%) DEGs were common DEGs respectively in drought and salt stresses in roots were analyzed for
between root and leaf tissues, but the number of common DEGs under GO analysis. Based on GO analysis, up-regulated DEGs were categorized
drought stress was only 302 (2.6%), that means the number of common into 3 groups of biological process (BP), molecular function (MF) and
DEGs in both tissues under salt stress was 4.2-fold higher than drought cellular component (CC).
stress (Fig. 1). It can be concluded that the number of common pathways The aforementioned groups, BP, MF and CC consisted of 17, 11 and
between roots and leaves induced by salt stress is more than those of 12 categories, respectively (Figs. 2 and 3). In both tissues, in response to
induced by drought stress. both stresses, for CC most enriched GO terms were cell (about 87%), cell
As mentioned above, the number of DEGs was up-regulated under part (about 87%) and organelle (about 65%), whereas the two highly
stress conditions in roots was higher than leaves that point to the most enriched ones for MF were binding (49%) and catalytic activity
important role of roots in abiotic SRMs in Arabidopsis. In some plants like (42%) (Figs. 2 and 3). Furthermore, for BP the highest frequency of DEGs
cotton it has been reported that the number of DEGs in leaves was higher function was belonged to cellular process (53%), metabolic process
than roots under salt stress [17], but there are some reports in other (50%) and response to stimulus (36%), respectively (Figs. 2 and 3),
plants which showed the number of up-regulated DEGs in roots were which agreement with results of other researches about microarray meta-
higher than leaves under drought stress [18]. It indicates that different analysis in plants in response to biotic and abiotic stresses [6, 20].
species may use different tolerance mechanisms in response to different In BP, the significant DEGs in cellular process (GO:0009987) were
stresses. The root is the first tissue to sense stress conditions and it may be assigned to cellular response to stimulus, regulation of cellular process,
explained why, in some plants like Arabidopsis, the number of genes multi-organism cellular process, cellular metabolic process, single-
involved in the stress in root tissue is higher than other tissues, but in organism cellular process and positive regulation of cellular process
some plants like cotton, probably, leaves have a central role in the control (supplementary 2). For the second group in BP, metabolic process
of water loss and hence, more genes involved in the stress in this tissue (GO:0008152), the significant ones were assigned to GO terms such as
[19]. According to their stress response mechanisms, plants may be single-organism metabolic process, cellular metabolic process, organic
divided into classes: (i) plants that root has a central role and (ii) plants substance metabolic process and catabolic process.
that the leaf has a central role. In BP group, for signaling (GO:0023052) and response to stimulus
The expression changes of DEGs was from 1.5 to 137-fold respect to (GO:0050896), the number of DEGs in each tissue in salt stress was
the control (supplementary 1). The percent of DEGs which up-regulated higher than drought stress. In signaling group, 9.5 and 9.7 percent of
more than 10-fold in response to drought stress in the root tissue (3.1%) DEGs in the leaf and 9.1 and 10.3 percent of DEGs in the root were up-
was more than the leaf tissue (1.6%), but it was reverse in the case of salt regulated in response to drought and salt stresses, respectively (Figs. 2
stress, in roots, 1.6% of DEGs up-regulated more than 10-fold, while it and 3). Gene ontology of up-regulated DEGs showed that signaling
was 2% for leaves (supplementary 1). These results indicate that the category included DEGs involvement in hormone-activated signaling
genes in leaves and roots have different expression patterns in response pathways, signal transduction, MAP kinase kinase activity, ATPase ac-
to salt and drought stresses. tivity, calcium-dependent protein serine/threonine kinase activity and
receptor signaling protein serine/threonine kinase activity. Phytohor-
3.2. Gene ontology mones such as abscisic acid, auxin, cytokinin and gibberellic acid play
important roles in different process of growth and development and also
Gene ontology (GO) enrichment analysis was conducted to detect the response to biotic and abiotic stresses in plants [21, 22]. In Chai et al
function of up-regulated DEGs in response to drought and salt stresses in (2019) study, Expression of AtNCED3 (nine-cis-epoxycarotenoid dioxy-
root and leaf tissues in Arabidopsis. A total of 1617 and 2854 DEGs genase) which is an important gene in ABA biosynthesis, increased about

Fig. 1. Venn diagram of differentially expressed genes in response to drought and salt stresses in leaves and roots of Arabidopsis thaliana which created by Venny 2.0.

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Fig. 2. Gene ontology analysis in leaves. Frequency of most representative biological process terms in Arabidopsis thaliana under salt and drought stresses. Gene
ontology analysis was made in the AgriGO platform (FDR ¼ 5%). More details in Supplementary 2.

4.6-fold in Arabidopsis plants after salt stress. Also, in our study salt stress up-regulated DEGs in both tissues. Majority of up-regulated DEGs
increased expression of this gene about 1.8-fold in leaf tissue of Arabi- (>46%) was belonged to binding category (GO:0005488) and more than
dopsis plant (supplementary 1). 40% was belonged to catalytic activity category (GO:0003824) in each
Another major part of up-regulated DEGs in BP group, was belonged tissue (Figs. 2 and 3). The results were coincidence with that obtained by
to response to stimulus category. Based on GO results, 37 and 38 percent Gao et al (2008) in response to drought stress in chickpea [23].
of DEGs in leaves and 31 and 36 percent of DEGs in roots were up- Some significant GO terms of binding group were ion binding, lipid
regulated in response to drought and salt stresses, respectively (Figs. 2 binding, cofactor binding and protein binding and significant GO terms
and 3). Components of this group were up-regulated in response to of catalytic activity group were oxidoreductase activity and hydrolase
abiotic stimulus, such as temperature, salt and drought stresses, response activity. Catalytic activity role in response to abiotic stresses especially
to endogenous stimulus such as phytohormones. osmotic stress was demonstrated [24]. Based on previous studies, anti-
Components of MF group have important roles in response to abiotic oxidant activity plays an important role in abiotic and even biotic stresses
stresses. In this category, catalytic activity and binding groups have more tolerance, but despite its important role in plant defense against osmotic

Fig. 3. Gene ontology analysis in roots. Frequency of most representative biological process terms in Arabidopsis thaliana under salt and drought stresses. Gene
ontology analysis made in the AgriGO platform (FDR ¼ 5%). More details in Supplementary 2.

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stress, amount of up-regulated DEGs in this group was low in each tissue groups. The greatest number of these TFs was belonged to NAC family
and in each stress, so that amount of this group was 1.4 and 0.94 percent followed by AP2/ERF, HSF, MYB, C2H2 and bZIP accounting for 68% of
in leaf and 0.85 and 1.1 percent in root respectively under drought and the total TFs (Fig. 5). Furthermore, common TFs in roots were divided
salt stresses. This kind of results has also been reported in earlier studies into 23 groups which the greatest number of them was belonged to MYB
[25]. Genes encoded antioxidant activity are one of the most important family followed by AP2/ERF, NAC, DOF, bZIP and HSF accounting for
genes in response to abiotic stresses. In plants during of abiotic and biotic 64% of the total TFs (Fig. 6).
stresses, reactive oxygen species (ROS) generated which severe damage AP2/ERF family is one of the most important transcription factors
to whole cells and organisms. Antioxidant enzymes such as superoxide family in plants which plays important roles in response to biotic and
dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), gluta- abiotic stresses [31, 32, 33]. In the present study, CRF7 (AT1G22985,
thione peroxidase (GPX), and glutathione reductase (GR) scavenge ROS CYTOKININ RESPONSE FACTOR 7), a member of ERF family, was
components in plants [25, 26]. Kanesaki et al (2002) identified which salt up-regulated in response to drought and salt stresses in leaves whereas its
stress caused to increase the expression of superoxide dismutase in Syn- expression in roots increased only under salt stress (supplementary 3).
echocystis. In our study, this gene up-regulated in response to drought and The analysis of Arabidopsis microarray data previously showed that the
salt stress in leaf tissue[27]. Furthermore, Ghaffari et al (2013) reported expression of CRF genes changed in response to abiotic stresses [34].
an increase of ascorbate peroxidase 2-like protein and Cu/Zn superoxide Overexpression of HARDY (AT2g36450) gene, encoding a member of this
dismutase in a tolerant line of sunflower roots in response to drought family, improved drought and salt tolerance in transgenic Trifolium
stress [28]. In our study, the expression of ascorbate peroxidase gene alexandrinum L [35]. Our microarray analysis results also showed that the
increased in root tissue in response to drought and salt stress. Based on expression of this gene up-regulated in roots in response to drought stress
results of Li et al (2012), osmotic stress using polyethylene glycol (PEG), (supplementary 3). RAP2.1 (AT1G46768) which up-regulated in
increased the expression of monodehydroascorbate reductase (MDHAR) response to both stresses in leaves (supplementary 3), encodes a member
in leaves of wild type tomato. MDHAR play important role in plant of the DREB subfamily. It has previously shown that drought and cold
antioxidant system by regeneration of ascorbate. In our study, the stresses induced expression of RAP2.1 gene through ABA-independent
expression of this gene also up-regulated in leaf tissue in response to salt pathway [36]. The expression of RAP2.1 gene is negatively regulated
stress. by cold and drought stresses in Arabidopsis. Moreover, RAP2.6
Transcription factors play important roles in plant responses to (AT1G43160) and DREB19 (AT2G38340) which encodes a member of
stresses via regulation of downstream genes by binding to cis-acting el- ERF and DREB subfamilies, respectively, up-regulated in roots in
ements in the promoter region of target genes [29]. The percent of these response to drought and salt stresses, whereas up-regulated only in
proteins were low in our study as previously reported by Khojasteh et al response to salt stress in leaves (supplementary 3). In addition, it has
(2018). been reported that the expression of these genes significantly
The results of GO analysis showed a similarity pattern of DEGs up-regulated under salt stress in transgenic plants [36]. It has been pre-
function for leaf and root tissues in response to drought and salt stresses viously reported that the expression of DREB2A (AT5G05410), a gene
but differences were in specific DEGs and number of significant DEGs in which encodes a member of DREB subfamily, increased in response to
each group between two stresses in each tissue (supplementary 2). In salt, drought and heat stresses [32, 37]. One of the earliest responses to
roots, approximately in most categories of BP and MF groups, amount of drought stress is activation of ERF genes such as ERF8 (AT1G53170)
up-regulated DEGs under salt stress were higher than drought stress, but which results in the induction of ethylene biosynthesis [38]. The
in the CC group, the amount of up-regulated DEGs was approximately expression of both of these genes, DREB2A and FRF8, was up-regulated in
equal under both stresses except for membrane, membrane part and response to salt and drought stress in leaves, whereas up-regulated only
organelle part categories which amount of up-regulated DEGs was higher under salt stress in roots (supplementary 3). Arabidopsis transgenic plants
in drought stress. Whereas in leaf tissue, the amount of up-regulated contain TaERF1 showed tolerance to different stresses such as drought,
DEGs in different categories of gene ontology was not so much salt and low-temperature stresses [39]. It has been previously shown that
different between two stresses. These results may indicate that roots ERF104 (AT5G61600) and ERF105 (AT5G51190) which in this study
respect to salt stress is more sensitive than drought stress. up-regulated in leaves under both stresses (supplementary 3), play a
critical role in response to high light and freezing stresses [40].
3.3. Identification of DEGs encoding transcription factors in Arabidopsis in Another important transcription factor family in plants is NAC family.
response to drought and salt stresses There are some reports that showed the members of this family has key
roles in response to biotic and abiotic stresses [41]. In Arabidopsis, 105
Transcription factors (TFs) play important roles in plant tolerance to redundant putative NAC genes were identified [42]. Our results showed
environmental stresses. These proteins as trans-acting elements bind to that NAC019 (AT1G52890), NAC02 (AT5G04410) and NAC072
cis elements in promoter region of various genes involved in different (AT4G27410, RD26) were up-regulated in leaves in response to both
stresses and as a result cause to activate or repress the expression of target stresses, while their expression increased in roots only under salt stress,
genes and subsequently increase the plants tolerance [30]. With regard to whereas NAC01 (AT1G01010) and NAC032 (AT1G77450) were
the important role of transcription factors in plant response to different up-regulated in roots in response to both stresses and NAC032 was
stresses, we further identified and compared the expression profiles of up-regulated only in response to salt stress in leaves. NAC047
these proteins in root and leaf tissues of Arabidopsis under drought and (AT3G04070) was up-regulated in both tissues in response to both
salt stresses. stresses (supplementary 3). It has been shown that the Introduction of
Based on the results, 109 (about 6.6%) and 223 (about 7.7%) genes in NAC019 in Arabidopsis plants increased the tolerance to drought, salt,
leaves and 234 (about 7.4%) and 396 (about 10.5%) genes in roots and low-temperature stresses in transgenic plants [43]. Different studies
respectively in response to drought and salt stress were identified as TFs showed that other members of this family such as OsNAC1, OsNAC2,
(Fig. 4). Identified TFs were classified in 28 and 34 groups in leaves, and OsNAC10 and AtNAC2 have important roles in various abiotic stresses
37 and 40 groups in roots in response to drought and salt stress, [44, 45]. Furthermore, it has been also demonstrated the role of some
respectively (supplementary 3). In each tissue under both stresses, the members of this family in ABA-dependent stress-signaling pathway [44,
greatest TF families were belonged to AP2/ERF, MYB and NAC families. 46].
Moreover, other important TF families such as bZIP, HSF, C2H2 and bZIP family is another large family of transcription factor in plants
WRKY were also detected. In addition, 54 and 98 TFs respectively in leaf and 75 members of this family was recognized in Arabidopsis [47].
and root tissues were identified as common TFs between drought and salt Microarray analysis of Arabidopsis in response to drought stress showed
stresses (supplementary 4). Common TFs in leaves were divided into 19 that the expression of bZIP44 (AT1G75390) increased significantly in

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Fig. 4. Venn diagram of differentially expressed genes encoding transcription factors in response to drought and salt stresses in leaves and roots of Arabidopsis thaliana
which created by Venny 2.0.

shoot tissue [48]. Our results indicated that its expression increased in between abiotic and biotic stress signaling. Furthermore, the expression
response to both stresses in roots, whereas in leaves, up-regulated only in of AtMYB108/BOS1 was induced in response to oxidative stress, drought
response to drought stress (supplementary 3). Moreover, in this study, and salt stresses [49]. Our work also showed that the expression of this
other members of bZIP family such as AtbZIP50/TGA7 (AT1G77920), gene increased in roots in response to both stresses whereas, in leaves
AtbZIP55/GBF3 (AT2G46270) were detected in response to drought and up-regulated only in response to salt stress (supplementary 3). The results
salt stresses in leaves, whereas AtbZIP56/HY5 (AT5G11260) of the study showed that the expression of AT5G60890 (AtMYB34)
up-regulated in response to both stresses only in roots and in leaves up-regulated in root tissue in response to both stresses, whereas in leaves,
up-regulated only under salt stress (supplementary 3). its expression increased only in response to salt stress. Up-regulation of
MYB and WRKY are two families of transcription factors involved in this gene was identified in Arabidopsis plant in Bhargava et al (2013)
response to abiotic stresses in plants [48]. For example, it has been shown study by microarray and RNA-Seq analysis.
that the expression of AtMYB108/BOS1 gene induced in response to It has been shown that there are 74 members for WRKY family in
oxidative stress, drought and salt stresses. AtMYB108 also involved in the Arabidopsis [51]. AtWRKY25 (AT2G30250), a member of WRKY family,
crosstalk between abiotic and biotic stresses [49]. AtMYB65 expresses under different abiotic stresses such as heat, drought, salt and
(AT3G11440) is a member of this family which involves in GA signaling oxidative stresses [52, 53, 54]. Here also, its expression increased in
in growth and flowering processes [50] and here, up-regulated in response to both stresses only in leaves but in roots, its expression
response to both stresses in roots (supplementary 3). AtMYB108 increased just in response to salt stress (supplementary 3). In other spe-
(AT3G06490) regulated filament elongation and anther dehiscence by JA cies, it has also reported that the members of these families have
and GA signaling pathways. AtMYB108 is involved in the crosstalk important roles in abiotic stress responses. OsWRKY23 (01g53260), as

Fig. 5. Common transcription factors in leaves of Arabidopsis thaliana which up-regulated in response to drought and salt stresses.

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Fig. 6. Common transcription factors in roots of Arabidopsis thaliana which up-regulated in response to drought and salt stresses.

member of these family in rice, involved in the regulation of resistance to HSFB2A had the highest outdegree in the network, so that 16 genes,
salt, ABA, H2O2, osmotic and dark stresses [55]. They showed that the RHL41, ZAT6, DREB2A, HSFA2, HSFA6B, HSFA6A, HSFA4A, HSFB2B,
overexpression of OsWRKY23 in Arabidopsis caused to accelerate the leaf HSFB3, HSF4, NAC046, NAC032, NAC087, ZF2, CZF1, ZF2 were directly
senescence in darkness. affected by this TF (Fig. 7, supplementary 6). This is the first report that
Another important transcription factor family is HSF family. There showed this protein is a hub TF in response to salt and drought stresses.
are 21 genes which encodes the members of this family in Arabidopsis DREB2A was affected by RHL41, HSFB2A, NAC06 and NAC096
[56]. Our results indicated that some members of HSF family, HSFA2, whereas 15 genes such as NAC019, RD26, HSFA4A, HSFA3, HSFA2, AT-
HSFB2B, HSFA4A, HSFA6A and HSFB2A in leaves and HSFA2, HSFB3, HSFB2B and etc. were affected by this TF (Fig. 7, supplementary 6). DREB
HSFA6B and HSFA3 in roots were up-regulated in response to both of (dehydration responsive element binding) is a subfamily of AP2/ERF
drought and salt stresses (supplementary 3). These results are in accord transcription factors family that regulate ABA-independent signal trans-
with what others reported. Overexpression of AtHSFA2 in Arabidopsis, duction pathway [61]. DREB2A has one AP2/ERF domain and includes
increased the tolerance of plants against abiotic stresses [57]. These DRE-binding proteins (DREBs) which binds to dehydration-responsive
transcription factors are regulated by other proteins, for example, the element/C-repeat (DRE/CRT) in promoter of abiotic stress-responsive
transcription of AtHSFA3 is regulated by DREB2A gene via binding to two genes and regulates their expression [32, 37]. It has been previously
DRE core elements of DREB2A in the AtHSFA3 promoter region [58]. shown that the expression of DREB2A was induced in response to
These TFs have important roles in abiotic stresses in other species, it has dehydration, salt and heat stresses (5, 32, 37). Based on the gene network
been shown that the expression of tomato HSFA3 and wheat HSF3 analysis, HSFB2A, RHL41, DREB2A, ERF104 and HY5 genes affected
increased thermotolerance of Arabidopsis transgenic plants [59, 60]. The ZAT6 expression (Fig. 7, supplementary 6). This TF activated the
result of this study showed that the transcription factors family of expression of some genes encoding TFs such as RD26, HSFA4A, ERF-1,
AP2/ERF, MYB, NAC, bZIP, HSF and WRKY may play important roles in WRKY25 and NAC047 (Fig. 7). ZAT6 belongs to C2H2 subfamily of
response to drought and salt stresses in Arabidopsis. Zinc-finger transcription factors family [62]. Based on the results of Liu et
al (2013), salt and osmotic stresses regulated ZAT6 expression in Arabi-
3.4. Gene network analysis dopsis. The expression of RHL41 directly activated by ERF104 and
HSFB2A genes, and affected 14 genes such as DREB2A, HSFA2, HSFA4A,
In this study, a network was generated between common TFs in leaves WRKY25, ZAT6 ERF-1 and etc (Fig. 7, supplementary 6).
and roots in response to drought and salt stresses in Arabidopsis, using RHL41 (AT5G59820) which also known as ZAT12 is a member of
STRING 10.5. Only 113 of 152 TFs had interactions (supplementary 5) C2H2 zinc finger transcription factors family [63]. The expression of
and so all the disconnected nodes in the network were removed. Between ZAT12 induced by light, low and high temperature, wounding, osmotic,
the TFs, seven of them, HSFA2, HSF4, MYB47, MYB32, NAC047, NF-YA5 salt and oxidative stresses [64, 65]. It has been also proved ZAT12
and AT1G76580 were common in both tissues and both stresses. Sub- involved in the up-regulation of WRKY25 in response to oxidative stress,
sequently, the TFs which had interactions together, were subjected to which is one of the genes in ROS signal transduction [66]. Based on our
Cytoscape v2.8.2 for more analysis. Based on the results of Cytoscape, a knowledge, there are no reports to indicate that, AT-HSFB2B and
total of 550 interactions (edges) were detected between 113 TFs (nodes) HSFA4A play important roles in salt and drought stresses, and this is the
(Fig. 7, supplementary 6). In this network, hub genes were identified first report to show these members of HSF family may play as hub pro-
based on their outdegree (the number of genes which was affected by the teins in these stresses.
same gene) and those that had an outdegree equal or more than 10 Network analysis interestingly showed that STZ (salt tolerance zinc
considered as a hub gene which includes HSFB2A (belongs to HSF fam- finger), a TF from C2H2 family, is affected by 17 other TFs which is the
ily), DREB2A (belongs to AP2/ERF family), ZAT6 (belongs to C2H2 highest number in this network, but has no effect on other TFs (outdegree
family), RHL41 (belongs to C2H2 family), HSFB2B (belongs to HSF equal zero) (Fig. 7, supplementary 6). It has been previously shown that
family) and HSFA4A (belongs to HSF family) (supplementary 6). These this protein acts as a transcription repressor to elevate abiotic stress
results indicated that the members of HSF, AP2/ERF and C2H2 families tolerance in plants [67]. Hence, it can be resulted that STZ has a critical
appear to play critical roles in salt and drought stress response role in stress tolerance mechanism in Arabidopsis and can be considered
mechanisms. as a suitable candidate to enhance salt and drought tolerance in crop

7
R. Ghorbani et al. Heliyon 5 (2019) e02614

Fig. 7. Network analysis of differentially expressed genes encoding transcription factors detected in salt and drought stresses. Network produced by STRING (version
10) with a confidence score >0.4, and visualized by Cytoscape 3.6.0. Node colors refer to the family of transcription factor. Node size corresponds to the number
of outdegrees.

plants through genetic engineering approach. Declarations

4. Conclusion Author contribution statement

Drought and salt stresses are two main groups of abiotic stresses Razieh Ghorbani: Performed the experiments; Analyzed and inter-
which affect agriculture productivity in worldwide. Furthermore, there preted the data; Contributed reagents, materials, analysis tools or data;
is a crosstalk between abiotic and biotic stresses in plants. Therefore, Wrote the paper.
identification of molecular mechanisms in response to them is helpful to Abbas Alemzadeh: Conceived and designed the experiments;
increase simultaneously tolerance to drought and salt stresses. To ach- Analyzed and interpreted the data.
ieve deeper understanding of molecular mechanisms in Arabidopsis and Hooman Razi: Contributed reagents, materials, analysis tools or data.
identification of responsive genes to drought and salt stresses, we
identified up-regulated DEGs in leaf and root tissues in response to these Funding statement
stresses using microarray data analysis approach. We found that there
are some differences between roots and leaves in terms of DEGs. This work was supported by Shiraz University.
Generally, the number of DEGs which up-regulated in response to
stresses in roots was 1.5-fold higher than leaves, especially for drought
stress, whereas it can be concluded that the number of common path- Competing interest statement
ways between roots and leaves induced by salt stress is more than those
of induced by drought stress. Various proteins involved in stress The authors declare no conflict of interest.
response mechanisms that transcription factors are most important of
them; among these kind of proteins, three families such as HSF, AP2/ Additional information
ERF and C2H2, appear to play critical roles in salt and drought stress
response mechanisms in Arabidopsis. In addition, the results of this study Supplementary content related to this article has been published
introducing some new proteins such as STZ, a member of AP2/ERF online at https://doi.org/10.1016/j.heliyon.2019.e02614.
family, which plays a critical role in stress tolerance mechanisms in
Arabidopsis and can be considered as a suitable candidate for increasing References
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