Analysis of Brassica napus dehydrins and their Co-Expression regulatory networks in relation to cold stress

Accepted Manuscript Analysis of Brassica napus dehydrins and their Co-Expression regulatory networks in relation to cold stress Khazar Edrisi Maryan, Habibollah Samizadeh Lahiji, Naser Farrokhi, Hassan Hasani Komeleh PII:

S1567-133X(18)30114-5

DOI:

https://doi.org/10.1016/j.gep.2018.10.002

Reference:

MODGEP 19038

To appear in:

Gene Expression Patterns

Received Date: 9 July 2018 Revised Date:

21 October 2018

Accepted Date: 22 October 2018

Please cite this article as: Maryan, K.E., Lahiji, H.S., Farrokhi, N., Komeleh, H.H., Analysis of Brassica napus dehydrins and their Co-Expression regulatory networks in relation to cold stress, Gene Expression Patterns (2018), doi: https://doi.org/10.1016/j.gep.2018.10.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Analysis of Brassica napus Dehydrins and their Co-Expression Regulatory Networks in Relation to Cold Stress Khazar Edrisi Maryan1, Habibollah Samizadeh Lahiji1*, Naser Farrokhi2*, Hassan Hasani

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Komeleh1 1. Department of Plant Biotechnology, Faculty of Agriculture, University of Guilan, Rasht, Iran. 2. Department of Cell and Molecular Biology, Faculty of Biological Sciences and Biotechnology, Shahid Beheshti University. G.C., Evin, Tehran, Iran.

Corresponding authors: Habibollah Samizadeh Lahij ([email protected]) & Naser Farrokhi

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([email protected])

Abstract:

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Dehydrins (DHNs) are plant specific cold and drought stress-responsive proteins that belong to late embryogenesis abundant (LEA) protein families. B. napus DHNs (BnDHNs) were computationally analyzed to establish gene regulatory- and protein-protein interaction networks. Promoter analyses suggested functionality of phytohormones in BnDHNs gene network.The relative expressions of some BnDHNs were analyzed using qRT-PCR in

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seedling leaves of both cold-tolerant (Zarfam) and -sensitive (Sari Gul) canola treated/untreated by cold. Our expression data were indicative of the importance of BnDHNs in cold tolerance in Zarfam. BnDHNs were classified into three classes according to the expression pattern. Moreover, expression of three BnDHN types, SKn (BnLEA10 and

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BnLEA18), YnKn (BnLEA90) and YnSKn (BnLEA104) were significantly high in the tolerant cultivar at 12 h of cold treatment. Our findings put forward the possibility of considering these genes as screening biomarker to determine cold-tolerant breeding lines; something

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that needs to be further corroborated. Furthermore, these genes may have some implications in developing such tolerant lines via transgenesis. Keywords: Promoter analysis, Phylogeny, Phytohormones, Regulatory gene co-expression network, Transcription factors.

1. Introduction Plant growth and distribution are greatly under the influence of temperature variations, diurnal changes and periodic alternation between day and night. Plants undertake drastic 1

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changes in their physiology and anatomy to acclimatize and withstand the imposed temperature stress. However, in severe cases, either sudden rapid change or great incline/decline in temperature may lead to wilting and death (Thomashow, 1999). The acclimation process is a rather complicated phenomenon that overhauls many signaling

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pathways including corresponding genes, proteins and metabolites (Chinnusamy et al. 2004). Some of known cold-associated and inducible proteins are dehydrins (DHNs), antifreeze proteins (AFPs), heat shock proteins (HSPs), cold-shock domain proteins (CSDPs) and enzymes such as alternative oxidases and desaturases in addition to reactive

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oxygen species generating/scavenging enzymes (Heidarvand and Maali Amiri, 2010). Often these genes are under hormonal control and depend on the perception of the hormonal

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signals in specific plant tissues.

Canola is relatively cold-tolerant, however, spring frost can severely damage or even kill canola seedlings (Fiebelkorn and Rahman, 2016). Here, we mostly focus on analysis of dehydrins in canola (Brassica napus L.), the second most abundant source of edible oil (Uppstrom, 1995), in response to cold stress treatment and the regulation of the transcription of dehydrins and their co-expressed gene by phytohormones. DHNs belong to

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group II late embryogenesis abundant (LEA) genes that have remained conserved during evolution (Liu et al. 2017). Their preservation suggests their pivotal roles in plant development (Lan et al. 2013). DHNs are a multigene family with 10 members in Arabidopsis (Choi et al. 1999; Hundertmark and Hindcha, 2008), 10 in soybean (Yamasaki

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et al. 2013), 8 in rice (Wang et al. 2007), 13 in barley (Tommasini et al. 2008), 11 in poplar (Liu et al. 2012) and 23 in canola. The progenitor diploid genomes of B. napus are ancient

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polyploids, and large-scale chromosome rearrangements have occurred since their evolution from a lower chromosome number progenitor (Schmidt et al. 2001). Chromosomal gene location and homology analyses of syntenic regions have revealed that BnLEA genes are phylogenetically related to other Brassicaceae species (B. rapa, B. oleracea, Arabidopsis), and they have experienced an extra whole-genome triplication (WGT) event (Cheng et al. 2014). DHN family expansion has generally occurred through tandem duplication events and whole-genome duplications (WGD) in poplar, Arabidopsis and rice (Cannon et al. 2004; Wang et al. 2007, Liu et al. 2012). In contrast, segmental duplications and WGD were considered as the main patterns of LEA gene expansion in B. napus and 2

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other Brassicaceae (Liang et al. 2016). In a review by Yu et al. (2016) on all 108 BnLEA genes in the Brassica database, it was revealed that nearly two thirds of the BnLEA genes are associated with segmental duplications. DHNs are considered as stress proteins involved in formation of plant protective reactions

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against dehydration. For instance, tobacco and Escherichia coli overexpressing Prunus mume dehydrin, PmLEAs, demonstrated enhanced tolerance to cold and drought (Bao et al. 2017). Similarly, E. coli expressing a Y3SK2-type DHN from Cucumis sativus, CsLEA11, demonstrated enhanced tolerance to heat and cold stress (Lv et al. 2017). Overexpression

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of 3 Physcomitrella DHNs in tobacco improved plant performance with respect to cold and osmotic stress (Agarwal et al. 2017). In another study, transgenic tomato overexpressing

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Solanum habrochaites dehydrins, ShFHN, showed tolerance to numbers of abiotic stresses (Liu et al. 2015). Furthermore, DHNs had reported to have a protective role for functionality of some proteins such as lactate dehydrogenase and malate dehydrogenase in response to environmental stresses (Drira et al. 2013, 2015; Yang et al. 2015; Zhou et al. 2017), and antimicrobial property (Agrawal et al. 2017). DHNs are also involved in scavenging of reactive oxygen species via inhibition of lipid peroxidation under cold stress through binding

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to metal ions (Mittler 2002; Hara et al. 2003; 2016; Haimi et al. 2017). DHNs are hydrophilic in nature due to containing charged and polar amino acids (GarayArroyo et al. 2000) and are characterized by three highly conserved motifs known as K, Y, and S segments. Amongst these segments, K motif is present in all plant lineages including

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non-vascular plants (Abedini et al. 2017). Different functional roles for each segment were suggested and confirmed; cryoprotection, antioxidant and chaperon activity for K-segment

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(Koag et al. 2009), probable capability of binding non-specifically to DNA and RNA by Ysegment due to sequence similarity to nucleotide-binding site of plant and bacterial chaperones (Close 1996, 1997; Lin et al. 2012; Hughes et al. 2013), to varieties of phospholipids (Koomajiman et al. 2007; Koag et al. 2009; Petersen et al. 2012), and phosphorylated S-segment functions as a nuclear localization signal (Brini et al. 2007a). The K-segment forms an amphiphilic α-helix that might be involved in protein and membrane stabilization under unfavorable environmental conditions (Hara et al. 2001; Allagulova et al. 2003; Eriksson et al. 2011; Petersen et al. 2012; Darira et al. 2013; Liu et al. 2017; Malik et al. 2017).The serine phosphorylated S-segment by protein kinases 3

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(Jensen et al. 1998), possibly improves dehydrin interactions with specific signal peptides followed by their translocation into the nucleus (Goday et al. 1994; Jiang and Wang, 2004) and probably promoting DHN binding ability to metal ions (Alsheikh et al. 2003; Hara et al. 2005, 2011). In addition to above three segments, DHNs also have a less conserved motif

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known as ɸ-segments that are interspersed between K-segments (Campbell and Close 1997). The number and order of these segments define the corresponding sub-classes, YnSKn, YnKn, SKn, Kn, and KnS (Garay-Arroyo et al. 2000; Rorat 2006). Dehydrin biosynthesis is greatly under the influence of both dehydration stresses (Danyluk et al. 1998;

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Kovacs et al. 2008) and hormone signaling. Allagulova et al. (2003) reported that the synthesis of basic YnSK2 dehydrins are induced by drought and ABA, but not by low accumulated in response to cold.

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temperatures while, acidic classes of dehydrins, KnS, SKn, and YnKn, are preferentially Many plants demonstrate a direct interrelationship between the level of dehydrins and cold stress (Houde 1992; Robertson et al. 1994; Sarhan et al, 1997; Levi et al. 1999; Lim et al. 1999; Wisniewski et al. 1999; Zhu et al. 2000, Davik et al., 2013). Here, in order to comprehend the molecular roles of dehydrins, co-expressed gene regulatory networks

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pertaining dehydrins were established and further confirmed by in silico means via analysis of cis-regulatory elements within promoters and the corresponding transcription factors, in addition to highlighting protein-protein interactions. Moreover, BnDHNs’ expression patterns may provide a response-predictor of varieties being tolerant or susceptible to cold stress.

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Accordingly, comparative expression pattern of dehydrin family in two Iranian canola cultivars, Sari Gul and Zarfam, spring and winter varieties respectively, were analyzed by

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Real-Time PCR in response to cold treatment. The probability of a relationship between protein motifs, promoter characteristics and hormonal signaling network in dehydrins gene expression was discussed.

2. Material and Methods 2.1. Gene network analysis Co-expressed genes of 23 BnDHNs were obtained from ATTEDII ver.9.2 (Obayashi et al. 2018) using eight Arabidopsis homologous sequences (Table 2) with mutual rank of 50 or lower. The co-expressed genes (209 genes) were further evaluated by Cytoscape ver.3.5.1 4

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(Shannon et al. 2003) to confirm the biological processes. The genes with the “Response to cold” annotation (GO-ID: 9409 and p-value: 1.3369E-18; 24 co-expressed genes) were selected to create protein-protein interaction in STRING (Szklarczyk et al. 2017). Panther database (Mi et al. 2016) was used to confirm gene ontology of co-expressed genes.

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Promoter analysis was conducted to isolate the putative elements for verification of the above network. Plant Promoter Database (Hieno et al. 2008) and PlantCARE (Lescot et al. 2002) was conducted for promoter analysis. PlantPAN (Chow et al. 2015) was used to find related transcription factors of each cis-element in BnDHN promoters.

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Amino acid sequences of 23 BnDHNs (75-259 amino acids) and 36 dehydrins of Brassicaceae were obtained from Uniprot (The UniProt Consortium, 2017), and submitted to

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Motif Analysis at MEME (Timothy et al. 2009) for de novo motif finding. MEME works on the basis of E-values, estimate of the number of motifs that is expected to be find by chance alone, to pinpoint to the real motifs (Timothy et al. 2009). Phylogenic tree was generated using MEGA6 software (Kumar et al. 2013).

2.2. Plant growth, treatment and sampling

Two B. napus cultivars, “Sari Gul” (cold sensitive) and “Zarfam” (cold- tolerant) were

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obtained from Seed and Plant Improvement Institute of Iran. Seeds were surface sterilized with ethanol (75% v/v) for 5 min followed by diluted commercial bleach (1:3 v/v) NaOCl for 10 min, rinsed three times with sterile distilled water. Germinated seeds were transferred to Hoagland solution (Hoagland and Arnon, 1950). Seedlings (14 day old) were exposed to

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decreasing temperature from 25°C to 4°C during 2 weeks. The plantlets were incubated for another two weeks at 4°C that followed by treatment at -4°C for 24 h. The seedlings were

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harvested at 0 (control), 3, 6, 9, 12 and 24 h exposure to -4°C (Fiebelkorn and Rahman, 2015). The seedlings were frozen immediately in liquid nitrogen, and stored at -70°C for further analysis.

2.3. Gene expression analysis Total RNA was isolated from cold-treated and control plants using RNX-Plus (CinnaGen Co., Iran) according to the manufacturer’s instructions. cDNA was synthesized using 200 ng RNA by Thermo Scientific RevertAid First Strand cDNA Synthesis kit (Germany). The qRTPCR experiments were conducted using SYBR Green qPCR Master Mix (BIORON, Germany) in CFX 96 Bio-RAD thermo cycler (Germany) in triplicates. Gene-specific primers 5

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(Table S2) were designed using Oligo primer analysis software (Rychlik, 2007). REST software (Pfaffle et al. 2002) was used for statistical analysis. Heatmap was generated by Heatmapper (Babicki et al. 2016).

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3. Results 3.1. Motif analysis

The subclasses/types of BnDHNs are presented in Table 1 and Figure 1. K-segment exists in all dehydrins and seven dehydrins “BnLEA89, BnLEA90, BnLEA104, BnLEA405,

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BnLEA106, BnLEA107 and BnLEA108” contain Y-segment (Figure 1). All dehydrins except BnLEA7, BnLEA8, BnLEA9, BnLEA18, BnLEA19, BnLEA20, BnLEA21, and BnLEA22

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possess His-related residues especially His-His; indicating strong affinity to metals ions such as Zn, Cu, Ni and Mn without phosphorylation. No dehydrins with “H-X3-H” domain were seen (Table 1).

Protein sequences were deduced for B. napus, B. oleracea, B. rapa, B. juncea, R. sativus, A. thaliana and S. parvula. Both K- and S- segments were found to be highly conserved between members of Brassicaceae DHNs. Based on the presence and numbers of k-, S-

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and Y- segments, DHNs were classified as SKn, KnS, Kn, YnKn and YnSKn (Figure1, Table 1). According to motif analysis of 36 dehydrins in Brassicaceae family, YnSKn (basic) and SKn (acidic) groups were the most abundant (Figure S1). Theoretical pIs of BnDHNs were between 4.95 and 9.4 (Table 1).

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Phylogenetic analysis revealed a monophyletic tree in Brassicaceae when O. sativa DHN was used as outgroups (Figure 2).

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3.2. Cis-regulatory elements of BnDHNs Following co-expression analysis, the promoter sequences of BnDHNs and their coexpressed genes were checked to define putative cis-regulatory elements by PlantCARE. Abiotic, biotic and seed development-related elements with different frequencies for each were predicted (Table 2, Figures 3 and S2). The common abiotic stress-related ciselements were ABRE (ABA-responsive elements), DRE (dehydration-responsive elements), HSE (heat shock-responsive elements) and LTR (low temperature responsive elements). Biotic stress-related elements were auxin, gibberellin, salicylic acid, ethylene and methyl jasmonate (MeJa)-responsive elements. Endosperm specific SKn-1expression element 6

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seems to be the only seed development-related motif. Some elements were unique to each group of DHNs (Table 2). Phytohormones-responsive cis-elements were the most common amongst BnDHNs and their co-expressed genes (Figure 3 and S2). All BnDHNs predicted to have ABA responsive element except BnLEA15 (KnS) that has

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auxin (TGA-element), MeJA (TGACG-motif) and ethylene (ERE) cis-elements, instead (Table 2). BnDHNs of SKn, Kn and YnKn contain different cis-element sequences for the same hormone; SKn and Kn types have GARE-motif, a gibberellin-responsive element, while “YnKn” type has “P-box” instead (Table 2). Due to the existence of ABRE, TGA-element and

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P-box BnLEA89 and BnLEA90 (YnKn) seem to be under the influence of abscisic acid, auxin and gibberellin, respectively. “YnSKn” and “SKn” seem to be regulated by phytohormone

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signaling, as all phytohormone-responsive elements are evident in promoter sequences (Table 2), except for SKn that lacks gibberllin-responsive element. The frequency of cisacting regulatory elements involved in phytohormone response in BnDHNs and their coexpressed genes are presented in Figure 3. 3.3. Protein- Protein Interaction (PPI)

In order to clarify the protein-protein interaction (PPI) network of dehydrins, all attributed

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transcription factors according to PlantPAN and dehydrin real co-expressed genes tagged as “Response to cold” based on Cytoscape, were used. Additionally, homologous sequences of BnDHNs in Arabidopsis were applied to STRING and the PPI network was obtained (Figure 4). Co-expressed genes were mainly involved in responding to cold stress

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within the cell through modulating the enzymes that are involved in metabolic pathways (Table S1). Low temperature-induced proteins (LTI65, LTI78) and cold-regulated proteins

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(COR15A, COR413-PMI and COR412IM1) were amongst interacting partners with homologous

sequences

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BnDHNs. Membrane

responses

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cold treatment were

limited and DREB1A, dehydration-responsive element-binding protein 1A, was the only transcription factor in PPI network (Table S1).

3.4. Relative gene expression analysis Quantitative RT-PCR analyses were carried out with eight genes belonging to the dehydrin subgroups that are known to be related in abiotic stress tolerance. Temporal expression of BnDHNs in two cold tolerant and sensitive cultivars are reported for 3, 6, 9, 12 and 24 h post 7

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treatment at -4 ºC. The expression data were classified into 3 groups. Group I (BnLEA7, BnLEA15, BnLEA88, BnLEA90 and BnLEA104) had the highest expression at 12 h with decline after 24 h in tolerant variety. BnLEA10 was the only representative in group II, which was significantly increased at 24 h in tolerant variety. Groups I and II remained significantly

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expressed in cold-sensitive variety. BnLEA18 and BnLEA67, were classified in group III, which exhibited increased expression at 12 h and their levels decreased at 24 h in tolerant variety. A delayed increase (at 24 h) in these DHNs was observed in the sensitive variety (Figure 5).

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By looking at the cis-regulatory elements of the genes with highly significant expression at 12 h cold acclimation, namely BnLEA10, BnLEA18, BnLEA90 and BnLEA104 (Figure 5), 9

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common elements were detected (Figure 6). Except for the common elements, CAAT-Box, TATA-Box and G-Box, the most related common cis-elements were TC-rich repeats (cisacting element involved in defense and stress responsiveness), ABRE (abscisic acid responsive element), Skn-1_motif (cis-acting regulatory element required for endosperm expression), and TCT-motif (part of a light responsive element). The presence of ABRE suggests that these genes are under the control of abscisic acid.

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4. Discussion

The involvement of DHNs in cold response have been demonstrated via transcript analysis in numbers of recent studies (Weiss and Egea-Cortines 2009; Yamasaki et al. 2013; Vaseva et al. 2014; Fu et al. 2016; Haimi et al. 2017; Zhou et al. 2017). In attempt to define BnDHNs

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co-expressed and regulatory gene networks, a rigorous bioinformatics approach was performed. Additionally, comparative gene expression analyses of canola dehydrins were

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carried out in cold sensitive/tolerant varieties. According to the expression patterns, the candidate dehydrins were classified into different groups. 4.1. Motif analysis

DHN molecular functions can be speculated according to the presence of particular segments in their sequences. All 23 BnDHNs contain K-segment (Table 1), and therefore may have cryoprotectant, antioxidant and chaperon activity (Koage et al. 2009). The numbers of K-segments fluctuate between 1 and 2 in BnDHNs, except for BnLEA88 that has 5 K-repeat motifs (Table 1). Functional analysis of a pepper dehydrin, CaDHN1, with 3 ksegments (SK3) revealed its involvement in abiotic stress tolerance with no significant 8

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response to either ABA or heavy metal treatments (Chen et al. 2015). As stated, DHNs may bind to nucleic acids via the Y-segment and here out of 23 BnDHNs only seven dehydrins have this motif (Table 1). KEKE motif was evident for 8 BnDHNs (Table 1) that suggests their potential to bind to nucleic acids via a zinc cation (Hara et al. 2005). Dehydrins are

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intracellular proteins, which accumulate to high levels in cell cytoplasm but they can also be found in several organelles (Brini et al. 2007a). All BnDHNs possess S-segment except “BnLEA67” that allows possible phosphorylation and nucleus localization (Brini et al. 2007a). However, nuclear localization has also been reported for dehydrin proteins lacking the S-

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segment such as WCS120 in cold-treated wheat crown tissue (Houde et al. 1995). For details of other cellular location of dehydrins see Graether and Boddington (2014). In

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addition to cellular localization, promoter analysis of a wheat dehydrin (DHN-5) via fusion with β-glucuronidase (gusA) gene in both Arabidopsis and wheat transgenic lines revealed organ-specific expression via GUS assay (Ben Amar et al. 2013). It has been proposed that dehydrins can bind to metal ions via histidine residues present in His-X3-His and His-His motif. BnDHNs, except eight (BnLEA7, BnLEA8, BnLEA9, BnLEA18, BnLEA19, BnLEA20, BnLEA21, BnLEA22; Table 1), have His residues mostly in H-H motif

corroborations.

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that suggest their capability of binding to metal ions; something that needs further

Phylogenetic tree looks monophyletic in Brassicaceae; with members of different genera appear within each phylum and it seems that segmental gene duplications and further

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genome rearrangements between species have occurred (Figure 2). Dehydrins being monophyletic was reported in Citrus sinensis L. Osb. (Pedrosa et al. 2015), and when a tree

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was generated that contained rice, Arabidopsis and potato (Charfeddine et al. 2017). Phylogenetic analysis of Pinaceae showed two clades; one only contained Picea taxon and the other contained a mixture of Picea and Pinus taxon (Sena et al. 2017).

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Promoter analysis

Identifying common cis-elements in promoter regions of the hub genes and their coexpressed gene network, verifies the likelihood of co-expression, and further allows defining the probable common regulatory trans-elements that control the corresponding network. In the study by Zolotarov and Strömvik (2015) in the analysis of promoter cis-elements, it 9

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became evident that promoter motifs are conserved across plant lineages for classes of dehydrins. All 23 BnDHN sequences were identified to contain DRE and ABRE sequences (Figure 3). DRE and ABRE are well known as cis-acting elements for stress-induction of many stress responsive genes (Baker et al. 1994; Yamaguchi-Shinozaki and Shinozaki

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1994; Jiang et al. 1996; Yamaguchi-Shinozaki and Shinozaki 2005). In addition to common cold-responsive elements, different cis-acting regulatory elements involved in phytohormone responsiveness were also identified in BnDHN promoter and their co-expressed genes (Table 2). Specific hormone signaling networks determine the induction of different groups

jasmonate and ethylene signaling network (Table 2).

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of dehydrins. For instance dehydrins of “KnS” group were under the control of auxin, methyl

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Plant hormones regulate every aspects of plant growth and development (Peleg and Blumwald, 2011) and are essential for the ability of adaption to abiotic stresses by mediating a wide range of adaptive responses (Santner and Estelle 2009; Wang et al. 2009; Messing et al. 2010). They often rapidly alter gene expression by inducing or preventing the degradation of transcriptional regulators (Santner and Estelle, 2009). The homeostasis of hormonal changes in response to cold follows a rapid response, adaptation to stress, and

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resistance stage. In rapid response also known as alarm phase, a surge in ABA (Yamasaki et al. 2013; Fu et al. 2016) is evident that coincide with down regulation of cytokinins, auxin and gibberlins (Kosova et al. 2012). Thus, in the first phase plant reprograms and manages itself towards survival rather than growth. Subsequently and after a few days of cold

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exposure, the hormonal balance will be reversed and content of other stress hormones (salicylic acid and jasmonic acid) will begin to increase. In a prolonged cold exposure, the

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cold tolerance will be enhanced and yet again the roles of phytohormones are undeniable through decline in cytokinins and auxin content in cold tolerant genotypes. In contrast and in the cold sensitive varieties, relatively higher cytokinin and auxin contents are being detected during acclimation period, and this most likely is related to the cause for reduced tolerance (Kosova et al. 2012). In addition to the mentioned hormones, brassinolides are also involved in cold signaling pathway through upregulation of few genes including a type II SSK2 dehydrin (Li et al. 2012). Diversity in cis-acting elements involved in auxin, gibberellin and MeJa responsiveness indicates the involvement of different transcription factor families in the expression of 10

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dehydrin genes (Table 2) as reported elsewhere (Choi et al. 1999; Zhu et al. 2000; Rodríguez et al. 2005; Tommasini et al. 2008; Yang et al. 2012; Ben Amar et al. 2013; Wang et al. 2014; Zhu et al. 2014; Lv et al. 2017). According to the dispersal of cis-acting

basic/acidic dehydrin expression and the hormone signaling.

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elements responsive to hormones, it seems that there is no correlation between the

In silico promoter analysis of 24 co-expressed genes of dehydrins introduced different common and specific cis-acting elements (Figures 3 and S2), illustrating the contribution of different hormone signaling similar to canola dehydrin gene expression networking. Auxin,

gene expression network in co-expressed genes.

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4.3. Protein-Protein Interaction network

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gibberellin, ABA and salicylic acid were the most abundant phytohormones in controlling

Protein-protein interactions (PPIs) play crucial roles in all biological processes. Generally, network analysis helps to understand a given biological system by unveiling interaction principles and patterns of functional cellular networks (Raman, 2010). Xie et al. (2012) demonstrated that Y2K2-type dehydrin of Medicago truncatula, MtCAS31, can interact with AtICE1 that is an inducer of CBF expression 1. This interaction in transgenic Arabidopsis

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thaliana led to reduced stomatal density and thus elevated drought tolerance. In recent studies and through in planta Bimolecular Fluorescence Complementation (BiFc) of DHNs, it was revealed that dehydrins tend to homo- and heterodimerize to become functional (Hernandez-Sanchez et al. 2014, 2017). Such interactions may suggest others that can be

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predicted based on the analysis of co-expression and dehydrins regulatory networks. The co-occurrence of proteins with dehydrins was illustrative of proteins mainly involved in

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carbohydrate metabolism and oxidative phosphorylation through Gol3 and RC12A, respectively. RC12A is an oxidoreductase localized to mitochondrion inner membrane as part of energy metabolism. PAP2.1, RD20 and Gol3 proteins with ion binding activity were the members of the signal transduction during cold stress. LTI65 and LTI78 (Nordin et al. 1993), low temperature induced proteins, and COR15A, COR413-PM1and COR412TM1 (Breton et al. 2003) were the other putative interacting proteins with dehydrins. The only transcription factor that appeared in dehydrin PPI network was DREB1A. DREB1A is a transcriptional activator that binds specifically to the DNA sequence [AG]CCGAC, C-repeat/DRE element, that mediates cold-inducible transcription 11

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(Sakuma et al. 2002). DREB1 factors play a key role in freezing tolerance and cold acclimation (Alonso-Blanco et al. 2005). In overexpression of soybean DREB1 in Arabidopsis, stronger up-regulation of downstream genes and better freezing tolerance were obtained (Yamasaki and Randall 2016).

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4.4. Gene expression

Real-time PCR analysis indicated increased amount of dehydrin gene expression during cold stress in tolerant canola genotype. It seems that all dehydrin groups were expressed in cold stressed B. napus (Figure 5). Three BnDHN types, Skn (BnLEA10 and BnLEA18), YnKn

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(BnLEA90) and YnSKn (BnLEA104) were strongly expressed at 12 h. This proves the importance of dehydrins in cell protection possibly through cryoprotection, antioxidant

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activity, and antifreeze activity.

DHNs are able to bind to lipid vesicles, maintaining membrane structure, and scavenge reactive oxygen species (ROS) during cold acclimation (Rorat, 2006). More specifically, SK3-type DHNs reported to be related to cold tolerance (Danyluk et al. 1994; Houde et al. 2004; Rorat 2006; Yin et al. 2006; Xing et al. 2011) and this possibly under the regulation of DREB1A, key transcription factor for cold tolerance (Seki et al. 2001; Thomashow et al.

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2001; Lee et al. 2005; Lee et al. 2013). DRE- and ABRE-like sequences are well-conserved in the promoter sequences of co-expressed genes of BnDHNs, supporting the role of DHNs in stress cold tolerance in B. napus.

At transcript level, there has been repeatedly reported a correlation between dehydrin

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accumulation and plant acquired cold tolerance in short-term (7 to 21 days) cold acclimation studies (Houde et al. 1992; Vítámvás et al. 2007; Kosová et al. 2008b; Holková et al. 2009).

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Thus, relative accumulation of selected dehydrin transcripts can be considered as a reliable marker of plant cold tolerance under controlled conditions (Kosová et al. 2014; Fu et al. 2016; Lv et al. 2017). We propose two SKn (BnLEA10 and BnLEA18), 1 YnKn (BnLEA90) and 1 YnSKn (BnLEA104) as diagnostics tool for differentiating cold tolerant B. napus genotypes from the cold sensitive varieties. Zolotarov and Strömvik (2015) in de novo analysis of promoter motifs of 350 dehydrin sequences found SKn and YnSKn dehydrins have elements connected with cold/dehydration, but not for YnKn. Earlier Vaseva et al (2014) proposed transcript analysis of 1 representative of each SKn (GenBank ID:

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EU846208) and YnSKn (GenBank Id: KC247804) to be served as diagnostic tool for differentiation of tolerant vs. sensitive genotypes of Trifolium repens.

5. Conclusion

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The results showed that there could be a relationship between protein motifs, promoter contents and hormonal signaling network in gene expression of canola dehydrins. Dehydrins were sorted to different groups according to amino acid motifs with specific characteristics (acidic or basic); discriminating specific protein function under the control of unique plant

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hormone signaling network with specific transcriptional factor families. PPI network presented the relationship between dehydrins, their transcriptional factors and co-expressed

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genes. Experimental analysis of gene expression emphasized the importance of dehydrins in response to cold stress in B. napus. Here, the novelty was due to the experiment setup: a time course at subzero temperatures followed by cold acclimation. Cold acclimation at 4 °C could be an explanation of the relatively low response of some of the dehydrins like XERO2 at -4 °C, assuming maximal response is being occurred at 4 °C after two weeks. We assume that responses are more of generic stress/damage response at - 4 °C. In line with

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earlier studies, we have found the most common cis-elements belong to hormonal responses. Accordingly, plant engineering towards hormonal biosynthesis can be envisaged for the production of more cold tolerant plants. However, the control of hormone dose/response ratio remains a challenge to have a proper balance between improved

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abiotic stress tolerance and the negative effects on growth and development (Peleg and Blumwald, 2011). The use of conditional promoters driving gene expression at specific

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developmental stages, in specific tissues/organs and/or in response to specific environmental cues circumvents this problem and will facilitate the generation of transgenic crops able to grow under various abiotic stresses with minimal yield losses (Peleg and Blumwald, 2011). Therefore, comparative analysis focused on the effects of phytohormone treatment on DHN gene expression during cold stress is suggested.

Acknowledgment The authors thank Iran National Science Foundation (INSF) (Grant number: 95814286) for financial support. 13

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Funding This work was supported by the Iran National Science Foundation (INSF) (Grant number:

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118.Zhu, B., Choi, D.W., Fenton, R., Close, T.J., 2000. Expression of the barley dehydrin multigene family and the

development

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of

five

classes

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dehydrin

Plos

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e0129016.

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10.1371/journal.pone.0129016.

genes.

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pI

BnaC07g15380D BnaAnng29030D BnaA07g11450D BnaC05g15780D BnaC03g44340D BnaA02g36030D BnaA07g32420D BnaA07g21490D BnaC06g21970D BnaC06g36880D BnaA07g12750D BnaA09g43150D BnaA09g31640D BnaC08g35660D BnaC08g22390D BnaCnng20790D BnaA01g05360D BnaC01g00220D BnaA02g34800D BnaA09g07980D BnaC02g45160D BnaC09g08130D BnaCnng27850D

216 194 259 271 95 194 184 184 184 184 75 183 128 128 128 245 149 149 190 144 189 186 144

4.95 5.62 5.07 5.09 6.7 5.61 5.51 5.47 5.47 5.44 9.4 6.67 9.36 6.38 9.19 6.75 5.5 5.89 7.14 7.14 7.97 7.14 7.14

K-

S-

Y-

Class/ KEKE Type motif

2 2 2 2 1 2 2 2 2 2 1 2 2 2 2 5 1 1 2 2 2 2 2

1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 0 1 1 1 1 1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 2 2 2 2 2

SKn SKn SKn SKn KnS SKn SKn SKn SKn SKn Kn SKn SKn SKn SKn SKn YnKn YnKn YnSKn YnSKn YnSKn YnSKn YnSKn

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Protein length(aa)*

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BnLEA7 BnLEA8 BnLEA9 BnLEA10 BnLEA15 BnLEA18 BnLEA19 BnLEA20 BnLEA21 BnLEA22 At3g50980 BnLEA67 (XERO1) BnLEA68 BnLEA69 BnLEA70 BnLEA71 At4g38410 (XERO2) BnLEA88 At4g39130 BnLEA89 BnLEA90 At5g66400 BnLEA104 (RAB18) BnLEA105 BnLEA106 BnLEA107 BnLEA108 Asterisks (*): Liang et al., 2016.

*

ID

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At1g20440 (COR47) At1g20450 (ERD10) At1g54410 (HIRD11) At1g76180 (ERD14)

*

Gene name*

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A.thaliana ID*

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Table 1: Summary and characteristics of B.napus DHNs: Gene name, ID, amino acid number, pI (isoelectric point), number of K-, S-, Y-segments, Class, KEKE motif and H-rich domain. DHNs mainly fall in 3 groups including Kn (BnLEA67), SKn (BnLEA69, BnLEA71) and YnSKn (BnLEA104, BnLEA105, BnLEA106, BnLEA107 and BnLEA108) with acidic residues. Seven DHNs, namely “BnLEA89, BnLEA90, BnLEA104, BnLEA105, BnLEA106, BnLEA107 and BnLEA108” contain Y-segment (Figure 1). All DHNs except BnLEA7, BnLEA8, BnLEA9, BnLEA18, BnLEA19, BnLEA20, BnLEA21 and BnLEA22 possess His-related residues especially His-His. These residues are indicators of strong affinity to metals such as Zn, Cu, Ni and Mn. No DHNs with “H-X3-H” domain was detected in B.napus.

1

1 1 1 1 1 1 1 1 -

H-rich domain H HH 10 0 9 0 11 0 13 1 14 2 9 0 6 0 6 0 6 0 6 0 5 1 13 4 8 3 8 3 8 3 21 3 9 1 10 1 9 3 9 3 9 3 8 3 9 3

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Table 2: Cis- regulatory elements in promoter regions of BnDHNs. Related transcription factors were obtained from PLANtPAN (http://PlantPAN.mbc.nctu.edu.tw/). DHN subgroups containing specific phytohormone cis-element are labeled with a star. Phytohormone cis-element Sequence Organism TF TF family KnS YnKn ABRE ACGTGGC Arabidopsis AT1G32150; AT2G35530 bZIP * Abscisic acid thaliana AuxRR-core GGTCCAT Nicotiana tabacum TGAAACGAC Brassica AT1G50680; AT1G51120 AP2;B3 * * element oleracea Auxin AuxRE TGTCTCAATAAG Glycine max P-box GACCAAACTCGT Pisum AT1G67260; AT2G31070; TCP * sativum AT3G02150 GARE-motif TCTGTTG Brassica oleracea Gibberellin TATC-box TATCCCA Oryza sativa AT3G16350 Myb/SANT TCACAGAAAAGGA Brassica AT2G28710; AT2G37430; C2H2, element oleracea AT3G46070; AT3G46080; WRKY Salicylic acid AT3G46090 CGTCACGTCA Hordeum AT1G77920; AT3G12250; bZIP motif vulgare AT5G06950; AT5G06960; AT5G10030; AT5G65210; AT1G22070 TGACGTGACG Hordeum AT1G08320; AT1G68640; bZIP * MeJA motif vulgare AT5G06839; AT5G10030 ERE ATTTCAAA Dianthus AT1G10480 C2H2 * Ethylene caryophyllus

2

YnSKn *

SKn *

Kn *

-

-

-

*

*

*

-

-

-

-

*

*

*

*

-

*

*

-

*

*

-

*

*

-

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Table 3: Gene ontology results of co-expressed genes using PANTHER. Category

Description/

ID

Biological process

Response to stimulus

GO:0050896

29.2%

70.0%

Cellular process

GO:0009987

4.2%

10.0%

Metabolic process

GO:0008152

8.3%

20.0%

Molecular

Binding

GO:0005488

8.3%

50.0%

function

Catalytic activity

GO:0003824

8.3%

50.0%

Cellular

Membrane

GO:0016020

4.2%

16.7%

component

Cell part

GO:0044464

20.8%

83.3%

Protein class

Transferase

PC00220

4.2%

25.0%

2*

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1*

Hydrolase

PC00121

4.2%

25.0%

Enzyme modulator

PC00095

8.3%

50.0%

*

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1 . The percent of genes classified to this category over the total number of genes (24 coexpressed genes). 2*. The percent of genes classified to this category over total number of class hits.

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Table S1: Accession number and biological process of 24 co-expressed genes with involvement in response to cold. GO-ID p-value Cluster frequency Total frequency 1.3369E-18

1.1604E-16 Genes

Gene name

Description

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Hydrophobic protein RCI2A HVA22-like protein e HVA22-like protein d Low-temperature-induced 65 kDa protein Galactinol synthase 3 Low-temperature-induced 78 kDa protein

Protein EARLY-RESPONSIVE TO DEHYDRATION- chloroplastic

RAB18 Cold-regulated 413 plasma membrane protein 1 Cold-regulated 413 inner membrane protein 1, chloroplastic Probable peroxygenase 3 Plasma membrane-associated cation-binding protein 1 AT3g17020/K14A17_14 Cysteine proteinase inhibitor 1 Dehydrin Xero 2 UPF0057 membrane protein At4g30650 Dehydrin COR47 Dehydrin ERD10 Ethylene-responsive transcription factor RAP2-1 Hydrophobic protein RCI2A HVA22-like protein e DREB1A,CBF3, CRAP2, ERF072, At4g25480, M7J2.150

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F5K20_290 RCI2A HVA22E HVA22D LTI65 GOLS3 RD29A ERD7 Dehydrin Rab18 COR413PM1 COR413IM1 PXG3 PCAP1 At3g17020 CYS1 XERO2 At4g30650 COR47 ERD10 RAP2-1 F5K20_290 RCI2A HVA22E Dehydration-responsive element-bind.

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Accession number AT3G53990 AT3G05880 AT5G50720 AT4G24960 AT5G52300 AT1G09350 AT5G52310 AT2G17840 AT5G66400 AT2G15970 AT1G29395 AT2G33380 AT4G20260 AT3G17020 AT5G12140 AT3G50970 AT4G30650 AT1G20440 AT1G20450 AT1G46768 AT3G53990 AT3G05880 AT5G50720 AT4G25480

241/22304 1.0%

RI PT

9409

4

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Table S2: Primer sets used for Real-time PCR

Reverse (5'-3') TCCATGTCATCCCAGTTGCT TCTGGAAGAGACTGGGCTTG TGGTTTTTCGCTGTGACCAG TTCAGTGCTCTTGGCGTGAT TTAATCGCTGTCGCTGTCAC AGCTAGAAGAGCTGTCGCTT GTGTTTAACCTCAGGCACCG TAAACGGGTATGGTGCTGGT CCGCCATTCTAATTAGC TCATCCCCTTCTTCTCGTGG

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TCCCGAGTATTGTTGGTCGT CGCTAGAGTCAGAGGTCGAG GGAGAACAAGCCCAGTCTCT GGTCACAGCGAGAAACCAGA GGTGAGCACAAGGAGGGTAT ACGGATCGAGGACTGTTTGA GCCACCGAGGAATCATCAAC CAGAAGGTGGTGGTGGTTTG CATGTATACACTACACAGAGCGC ATGATGGACAAGGTGGGAGG

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ACTIN BnLEA7 BnLEA9 BnLEA10 BnLEA15 BnLEA18 BnLEA67 BnLEA68 BnLEA90 BnLEA104

Forward(5'-3')

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Gene name

5

TM (°C) 58.4 59.35 57.30 56 57.30 57.30 59.35 57.30 60.56 59.35

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Figure 1: Conserved motifs in 23 B.napus DHN. The motifs were found by MEME (http://meme suite.org/). Motifs 2 (S-segment: EEEEEENKPSLLEKLHRSGSSSSSSSEEE, light blue), 3 (K-segment: EEEEKKGMMEKIKEKLPGHG, light green) and 7 (Y-segment: DEDGNPVGTTAAVPV, dark blue) are part of BnDHN domain structure. Motif identifiers (color boxes with numbers 1 to 15) are described in Supplementary Figure 1. The lengths of the proteins and motifs can be estimated using the scale at the bottom.

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Figure 2. Phylogenetic analysis of DHNs in Brassicaceae. The tree was constructed in MEGA6 using maximum parsimony method based on a full-length amino acid sequence alignment of DHNs from Brassicaceae (B. napus, B. oleracea, B. rapa, B. juncea, R. sativus, A. thaliana, S. parvula) and O.sativa was used as outgroup.

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25

20

15

10

cis-elements

number of cis-elements in dehydrin co-expressed genes

CAAT-box G-Box Unnamed__4 ABRE Unnamed__3 TCT-motif AAGAA-motif LTR circadian Box I GCN4_motif GATA-motif ERE HSE O2-site box S GARE-motif AE-box TATCCAT/C-motif ACE W box ATCT-motif CGTCA-motif AT1-motif Unnamed__2 CAT-box A-box 3-AF3 binding site Unnamed__5 GA-motif TCCC-motif Unnamed__11 RY-element CTAG-motif 3-AF1 binding site Unnamed__13 ATC-motif box E C-repeat/DRE Box III Unnamed__6 AC-I E2Fa chs-CMA1a ATCC-motif TATC-box Unnamed__10 Unnamed__14 chs-CMA2c chs-Unit 1 m1 as1 HD-Zip 2 CAG-motif Unnamed__16

5

0

Number of cis-elements in dehydrin

3

Figure 3: Identification and the frequency of each cis-element in BnDHNs and their co-expressed genes (For information about coexpressed genes, see Table S1). PlantCARE: http://bioinformatics.psb.ugent.be/webtools/PlantCARE/html PlantPAN (http://PlantPAN.mbc.nctu.edu.tw/)

Frequency of cis-elements

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Figure 4: Protein-protein interaction network of BnDHNs, transcription factors and co-expressed genes identified using STRING. For TFs and co-expressed gene list refer to Supplementary Table 1. LTI65, LTI78, COR15A, COR413-PMI, COR412IM1, DREB1A, PAP2.1, RD20 proteins are among the cluster. Dehydrin co-expressed genes were involved in different metabolic pathways including carbohydrate metabolism and oxidative phosphorylation through Gol3 and RC12A, respectively.

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20 15

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Relative expression

25

10 5

z

s

BnLEA7(COR47)

z

s

BnLEA10(ERD10)

z

s

z

BnLEA15(HIRD11)

s

z

BnLEA18(ERD14)

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9h

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(b)

s

BnLEA67(XERO1)

Canola varieties 3h

SC

0

5

12h

z

s

BnLEA88(XERO2-like)

z

s BnLEA90

z

s

BnLEA7104(RAB18)

24h

(a) Figure 5: Relative gene expression profile. (a) The results of quantitative RT-PCR relative gene expression levels of nine DHNs in two canola varieties (Zarfam (Z) and Sari Gul(S)) during 3, 6, 9, 12 and 24 h post cold treatment. REST software (Pfaffle et al. 2002) was used for statistical analysis. Error bars represent standard deviation. Actin was used as reference gene to normalize the expression values. (b) Heat map was performed to demonstrate hierarchical clustering of the expression pattern of DHNs during the analyzed time periods. The color scale represents relative expression levels with increased (green) or decreased transcripts (red). Interestingly, DHNs show pronounced expression in cold-tolerant variety (Zarfam) and more specifically at 12 and 24 h post cold treatment.

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Figure 6: Venn diagram of cis-regulatory elements of 4 highly expressed BnLEAs after 12 h of cold acclimation (Figure 5). The diagram was generated by Venny 2.1 (Oliveros 2007).

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BnLEA10, BnLEA18, BnLEA90, and BnLEA104 had 9 common cis-elements:

CAAT-Box,

TATA-Box, 2 G-Boxes, TC-rich repeats (cis-acting element involved in defense and stress responsiveness), ABRE (Abscisic acid responsive element), Skn-1_motif (cis-acting regulatory element required for endosperm expression), TCT-motif (part of a light responsive element) and an unnamed motif.

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5%3%

YnSkn

8%

SKn Kn 27%

57% KnS

a. E-value

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b.

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Gene Name

RI PT

YnKn

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Figure S1: a: The frequency of DHN subclasses (YnSKn, YnKn, SKn, Kn, and KnS) in Brassicaceae (36 members). b: Conserved motif analysis of DHN subclasses (YnSKn, YnKn, SKn, Kn, and KnS) in Brassicaceae (36 members) by MEME. Motif 2 (S-segment), 3 (K-segment) and 7 (Y-segment) are part of BnDHN domain structure in Brassicaceae. The lengths of the proteins and motifs can be estimated using the scale at the bottom.

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Ethylene

RI PT

MeJA Salicylic acid Gibberellin

ABA 0

5

10

15

20

25

30

35

Dehydrins

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Coexpressed genes of dehydrins

SC

Auxin

Figure S2: The number of cis-regulatory elements involved in phytohormone responsiveness in promoter

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region of BnDHNs and their co-expressed genes (For information about co-expressed genes, see Table S1).

8