Pramod Rathor
email: pramod.rathor@dal.ca

Tudor Borza
email: tudor.borza@dal.ca

Ramin Bahmani
email: rbahmani@dal.ca

Sophia Stone
email: s.stone@dal.ca

Thierry Tonon
email: thierry.tonon@york.ac.uk

Svetlana Yurgel
email: syurgel@dal.ca

Philippe Potin
email: philippe.potin@sb-roscoff.fr

Balakrishnan Prithiviraj
email: bprithiviraj@dal.ca

Expression of a Heat Shock Protein 70 from the Brown Alga Ectocarpus sp. Imparts Salinity Stress Tolerance in Arabidopsis thaliana

Keywords: Arabidopsis thaliana, Ectocarpus sp., Phaeophyceae, heat shock protein, salinity

The brown alga Ectocarpus sp. belongs to Phaeophyceae, a class of macroalgae in the phylum Heterokonts, which is also known as the Stramenopiles lineage. Ectocarpus is a dominant seaweed in temperate regions around the globe, abundant mostly in the intertidal zones, a habitat with harsh environmental conditions resulting from tidal cycles. Analysis of previously generated transcriptomic data of brown alga showed the up-regulation of the Esi0379_0027 gene, encoding a HSP70 protein, in response to various abiotic stresses. Bioinformatics study demonstrated that the HSP70 protein is soluble, monomeric and well conserved compared to other organisms. Subcellular localization of EsHSP70 revealed that the protein is localized in nucleus. To explore the role of HSP70 in stress tolerance, it was expressed in Arabidopsis under constitutive and stress inducible promoters. Transgenic Arabidopsis plants generated using both promoters exhibited higher tolerance to salinity stress compared to wild type plant. Transcript analysis of various abiotic stress-responsive genes showed that genes participating in Na + efflux and sequestration, and in ABA-mediated stress tolerance were remarkably upregulated in the EsHSP70-expressing transgenic Arabidopsis. Overall, our results showed that the expression of the EsHSP70 promotes salt stress tolerance by upregulating stress responsive genes in Arabidopsis.

Introduction

Plants being sessile organisms are incapable to avoid stressful or unfavorable growth conditions by changing place. These organisms are constantly exposed to several environmental stresses such as salinity, drought, high temperature, heavy metal and ultra-violet radiation. These stressors negatively affect plant growth and development leading to significant loss in productivity. High soil salinity is the predominant among various abiotic stresses which causes severe crop loss globally. In addition, the effect of global climate change further exasperates the effects of abiotic stresses on agricultural production [START_REF] Lesk | Influence of extreme weather disasters on global crop production[END_REF]. The crop loss due to these stressors account up to 50% loss in production [START_REF] Lobell | Climate trends and global crop production since 1980[END_REF]. High salinity inhibits plant growth in multiple ways such as osmotic stress, ion accumulation and secondary stress. The primary stresses, such as salinity, drought and high temperature are often interconnected and lead to secondary stresses such as oxidative stress. At cellular level, these stresses result in physiological dehydration, which further damages cellular machinery [START_REF] Wang | Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance[END_REF]).

Increased soil osmotic potential due to presence of salt reduces the plants ability to uptake water and nutrients [START_REF] Hasegawa | Plant cellular and molecular responses to high salinity[END_REF]. Accumulation of Na + and Cl -ions in significantly higher concentration than those of outside in the soil helps to maintain turgor pressure but cells must strictly regulate contents of these ions in each cellular compartment to avoid inactivation of enzymes thus prevent metabolic dysfunction [START_REF] Munns | Salinity tolerance of crops-what is the cost? New phytologist[END_REF]. Based on their ability to tolerate salt stress, plants are classified as halophytes and glycophytes [START_REF] Flowers | The mechanism of salt tolerance in halophytes[END_REF]; halophytes are highly tolerant to salinity stress. These plants have evolved unique structural features such salt secretory glands in leaves that has the capacity to excrete excess salt that ensures their survival under higher levels of soil salinity. However, most of the crop plants are glycophytic and lacks such structures therefore doesn't exhibit higher level of salinity stress tolerance [START_REF] Hasegawa | Plant cellular and molecular responses to high salinity[END_REF][START_REF] Julkowska | Tuning plant signaling and growth to survive salt[END_REF][START_REF] Hasegawa | Plant cellular and molecular responses to high salinity[END_REF].

Generally, halophytes have higher concentration of Na + and Cl -ions in leaves than the outside environment that helps to maintain higher osmotic potential in the cells and therefore higher turgor pressure. These plants store Na + and Cl -in vacuoles and uses compatible solutes and K + ions to equilibrate the osmotic differences in different compartments of cytoplasm [START_REF] Shabala | Learning from halophytes: physiological basis and strategies to improve abiotic stress tolerance in crops[END_REF]. Accumulation of Na + and Cl -ions in some leaves may be deleterious but at whole plant level it might be beneficial as plant can store the excess Na + and Cl -ions in those leaves and later remove these ions via abscission [START_REF] Munns | Mechanisms of salinity tolerance[END_REF].

Heat shock proteins are classified on the basis of their molecular weight into five major groups named as sHSP (small heat shock proteins), HSP60, HSP70, HSP90 and HSP100 [START_REF] Wang | Role of plant heatshock proteins and molecular chaperones in the abiotic stress response[END_REF]). These proteins are present and conserved in almost all living organisms from prokaryotes to eukaryotes (De [START_REF] Maio | Heat shock proteins: facts, thoughts, and dreams[END_REF]. Heat shock proteins have a vital role in developmental processes and response to abiotic stresses like salt [START_REF] Zou | Overexpression of OsHsp17.0 and OsHsp23.7 enhances drought and salt tolerance in rice[END_REF], drought [START_REF] Cho | A nuclear-localized HSP70 confers thermoprotective activity and drought-stress tolerance on plants[END_REF] and extremes of temperature [START_REF] Lopez-Matas | Protein cryoprotective activity of a cytosolic small heat shock protein that accumulates constitutively in chestnut stems and is up-regulated by low and high temperatures[END_REF]). Among several HSPs, HSP70 is a highly conserved protein and has been extensively studied in bacteria, plants and animals. It is the most abundant molecular chaperon, and is localized to various cellular compartments including cytosol, nucleus, mitochondria and endoplasmic reticulum, protecting proteins by folding of misfolded or unfolded proteins, disassembly of protein complex to translocate in another cellular compartment and in disaggregation of complex aggregates [START_REF] Saibil | Chaperone machines for protein folding, unfolding and disaggregation[END_REF]. HSP70 contains two main domains: a substrate binding domain and an ATPase domain [START_REF] Kampinga | The HSP70 chaperone machinery: J proteins as drivers of functional specificity[END_REF][START_REF] Mayer | Gymnastics of molecular chaperones[END_REF][START_REF] Zuiderweg | Allostery in the Hsp70 chaperone proteins. In: Molecular Chaperones[END_REF]. The activity of HSP70 depends on the interaction between these two domains and other co-chaperons [START_REF] Saibil | Chaperone machines for protein folding, unfolding and disaggregation[END_REF]. Proteins which are not folded correctly in their native state contains several binding sites usually at every 30 to 40 residues for HSP70 [START_REF] Rüdiger | Interaction of Hsp70 chaperones with substrates[END_REF]. It is known that HSP70 binds to the 7-residue segment of a polypeptide chain which is hydrophobic in nature [START_REF] Clerico | How HSP70 molecular machines interact with their substrates to mediate diverse physiological functions[END_REF]. Even transient binding of polypeptide segment with HSP70 can prevent the misfolding and aggregation of proteins thus maintains protein in native state [START_REF] Young | Mechanisms of the Hsp70 chaperone system[END_REF]. Once reached to right destination polypeptide regains the native state in free solution by detaching from HSP70. If it fails to refold properly it will lead to rebinding to HSP70. Therefore, the function of HSP70 in proper folding seems to be in stabilization of unfolded proteins until it can refold correctly [START_REF] Sharma | The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase[END_REF]). HSP70 proteins have been identified in several plant species [START_REF] Vierling | The roles of heat shock proteins in plants[END_REF][START_REF] Boston | Molecular chaperones and protein folding in plants[END_REF]. In Arabidopsis 18 genes encoding the HSP70 family proteins have been discovered. Analysis of the expression profile of these genes showed that members of HSP70 family were expressed in response to several environmental stimuli such as high temperature, cold, drought, salinity and other chemical stresses [START_REF] Lin | Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana[END_REF][START_REF] Sung | Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family[END_REF]. Several studies have been carried out and reported that overexpression of HSP70 improved drought, salinity and high temperature stress tolerance in transgenic plants. [START_REF] Cho | A nuclear-localized HSP70 confers thermoprotective activity and drought-stress tolerance on plants[END_REF], reported that constitutive overexpression of nuclear localized HSP70 from Nicotiana tabacum improved high temperature and drought stress tolerance in transgenic tobacco, whereas the antisense plants were highly susceptible [START_REF] Cho | A nuclear-localized HSP70 confers thermoprotective activity and drought-stress tolerance on plants[END_REF]. Similarly, constitutive expression of HSP70 from cotton improved drought stress tolerance in transgenic tobacco [START_REF] Ni | The cotton 70-kDa heat shock protein GhHSP70-26 plays a positive role in the drought stress response[END_REF]. Overexpression of HSP70 from chrysanthemum have shown to improve salinity, drought and high temperature stress tolerance in transgenic Arabidopsis [START_REF] Song | A chrysanthemum heat shock protein confers tolerance to abiotic stress[END_REF]. Arabidopsis plants transformed using fungal HSP70 from Trichoderma improved tolerance to heat, salinity, osmotic and oxidative stress [START_REF] Montero-Barrientos | Transgenic expression of the Trichoderma harzianum hsp70 gene increases Arabidopsis resistance to heat and other abiotic stresses[END_REF].

Seaweeds are an integral component of marine coastal ecosystems, where they form large dense forests and provides habitat for many organisms [START_REF] Khan | Seaweed extracts as biostimulants of plant growth and development[END_REF]. Brown alga are multicellular organisms belonging to Stramenopiles lineage (also known as Heterokonts) [START_REF] Cock | The Ectocarpus genome and brown algal genomics: The Ectocarpus Genome Consortium[END_REF]). This lineage is distantly related to Archaeplastida (including green plants and red algae) and Opisthokonts (including animal and fungi) [START_REF] Baldauf | The deep roots of eukaryotes[END_REF][START_REF] Brodie | The algal revolution[END_REF]. Since each of these lineages have evolved independently it is thought that organisms in each of these lineages have evolved mechanisms necessary for the development of complex multicellularity.

These mechanisms are well studied in land plants and in animals but very little is known in brown algae [START_REF] Coelho | Ectocarpus: A model organism for the brown algae[END_REF]. During the billions year of evolution brown algae have made the transition from unicellularity to complex multicellularity and therefore acquired novel features which are absent in other lineages [START_REF] Cock | The Ectocarpus genome and brown algal genomics: The Ectocarpus Genome Consortium[END_REF][START_REF] Brodie | The algal revolution[END_REF]. Endosymbiotic gene transfer led to the significant incorporation of foreign genes and enriched the nuclear genome. Some of these genes were targeted to specific organelle through N-terminal transit peptide and many of these evolved novel functions in these organisms [START_REF] Brodie | The algal revolution[END_REF]).

These novel features make it very interesting to explore and decipher the metabolisms and cellular biology of these organisms. Moreover, genome sequencing demonstrated that more than 36% of genes are of unknown functions and doesn't match to any other existing organisms. This indicates that these are novel genes or got considerably diverged from the homologue genes present in other organisms [START_REF] Cock | The Ectocarpus genome and brown algal genomics: The Ectocarpus Genome Consortium[END_REF]. Some of the novel features include metabolism of halogenated compounds, accumulation of complex polysaccharides and higher tolerance to biotic and abiotic stresses [START_REF] Charrier | Development and physiology of the brown alga Ectocarpus siliculosus: two centuries of research[END_REF]. Moreover, in brown algae the photosynthetically fixed carbon is not stored as starch granules, instead it is stored as laminarin and mannitol [START_REF] Craigie | Storage products[END_REF][START_REF] Davis | A review of the biochemistry of heavy metal biosorption by brown algae[END_REF]. In contrast to land plants, these organisms produce complex polysaccharides such as alginates and sulfated fucans [START_REF] Kloareg | Structure of the cell walls of marine algae and ecophysiological functions of the matrix polysaccharides[END_REF]. Genome editing tools have been routinely applied to land plants to understand their complex organization.

However, their application in other lineages for instance in Stramenopile is very challenging as these are not easily amenable, lack of standardized protocols for genetic transformation, mutation, promoter analysis and subcellular localization. The genome sequencing of Ectocarpus provided some insights to brown algal biology, however, various biological aspects still remains largely unknown. Transcriptomics study on brown algae showed that the expression level of several genes was upregulated under different abiotic stresses conditions, along with elevated levels of stress tolerance [START_REF] Ritter | Transcriptomic and metabolomic analysis of copper stress acclimation in Ectocarpus siliculosus highlights signaling and tolerance mechanisms in brown algae[END_REF]. One of these genes was identified as EsHSP70 (Esi0379_0027). In the present work, to examine the role of EsHSP70 in abiotic stress tolerance, it was expressed in A. rabidopsis thaliana. Transgenic Arabidopsis plants expressing EsHSP70 showed higher tolerance to salt stress. Furthermore, the transcript level of various stressresponsive genes was found to be significantly higher in HSP70-expressing transgenic plants compared to the control (normal condition without salt) plants.

Materials and Methods

Brown alga growth condition and RNA isolation

The axenic culture of brown algal model Ectocarpus sp. (Dilwyn) Lyngbye unialgal strain 32 (accession CCAP 1310/4, origin San Juan de Marcona, Peru) was established in a 10 liters L plastic tank filled with filtered, autoclaved natural sea water containing 1% (v/v) of provasoli Provasoli nutrient medium. Culture was maintained at 14 °C in the growth room with light cycle of 14h light/10h dark, with light intensity of 40 μmol. photons m -2 s -1 . Compressed air passing through a filter was used to continuously air bubble the nutrient medium. The culture was then treated with 1450 mM NaCl for 6 h to induce salt stress condition. Following the exposure to high salinity stress condition culture was collected using filtration, carefully dried on absorbent paper and promptly flash frozen in liquid nitrogen. Total RNA isolation was performed using 0.1 g of tissue following the protocol described earlier with minor modifications [START_REF] Apt | Evolution of the phycobiliproteins[END_REF].

The modifications to the protocol were made as previously stated [START_REF] Bail | Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus[END_REF]. One microgram of RNA was treated with Turbo DNAse (Ambion Austin, USA) following to the manufacturer's instruction. The DNAse-treated RNA was converted to cDNA, using a SuperScript IV Reverse Transcriptase (Life Technologies, France), following the manufacturer's procedures.

Bioinformatics analysis

The protein transmembrane helices or domain were identified using HMMTOP v.2.0 (http://www.enzim.hu/hmmtop/) and TMHMM v.2.0 (https://services.healthtech.dtu.dk/) prediction servers. To determine the subcellular localization of protein, the amino acid sequence was analyzed using TargetP 2.0, Signal P and DeepLoc-1.0 (https://services.healthtech.dtu.dk/), iPSORT (http://ipsort.hgc.jp), and WoLF PSORT (https://wolfpsort.hgc.jp) prediction servers.

Homology modelling for secondary structure was carried out using PSIPRED (Buchan and Jones 2019) (http://bioinf.cs.ucl.ac.uk/psipred). To obtain information about the tertiary structure and the folding of EsHSP70, protein model was generated using SWISS-MODEL [START_REF] Waterhouse | SWISS-MODEL: homology modelling of protein structures and complexes[END_REF]). The best model (77.64 % sequence identity) was built using bovine heat shock cognate 71 kDA as a template (SMTL ID: 4fl9.1; structure solved by Xray diffraction 1.90 Å). BlastP search was performed using EsHSP70 as a query to find the protein sequence homology with different other taxa. MUSCLE alignments [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]) of some selected protein sequences were done using MEGA X software [START_REF] Kumar | MEGA X: molecular evolutionary genetics analysis across computing platforms[END_REF]). The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model [START_REF] Jones | The rapid generation of mutation data matrices from protein sequences[END_REF]). The evolutionary analysis, comprised of 49 sequences including EsHSP70, analyses were performed using MEGA X software [START_REF] Kumar | MEGA X: molecular evolutionary genetics analysis across computing platforms[END_REF].

Plasmid construction and transformation of Arabidopsis

To create the plant transformation constructs, pEarleyGate 100 [START_REF] Earley | Gateway-compatible vectors for plant functional genomics and proteomics[END_REF]) and a promoterless vector pMCS:GW [START_REF] Michniewicz | Gateway-compatible tissuespecific vectors for plant transformation[END_REF] were obtained from ABRC (Columbus, OH, USA). Promoter sequence for a stress inducible gene RESPONSIVE TO DESICCATION 29A (RD29A) was amplified from the wild type (Col-0) Arabidopsis. Gateway vector containing stress inducible promoter of RD29A gene, was generated as described earlier [START_REF] Rathor | A novel protein from Ectocarpus sp. improves salinity and high temperature stress tolerance in Arabidopsis thaliana[END_REF]. The coding sequence of EsHSP70 was amplified by RT-PCR using gene specific primers flanked by attB sequences and cloned into pDONR221 by the BP Clonase TM II Gateway ® , following the manufacturer's protocol to generate entry clone (Gateway ® Technology with Clonase II, Invitrogen, Ontario, Canada). The entry clone harboring full gene sequence was then recombined into pEarleyGate 100 (35S) and pMCS:GW (RD29A promoter) by LR Clonase TM II Gateway ® , following the manufacturer's protocol (Gateway ® Technology, Invitrogen, Ontario, Canada). The correctness of the RD29A promoter and EsHSP70 sequences was confirmed by PCR using the forward primer sequence from the RD29A promoter and reverse primer sequence from the EsHSP70. The expression clone was then transformed into Agrobacterium strain GV310 (pMB90) using the freeze-thaw method followed by Arabidopsis (ecotype Col-0) transformation through the floral dipping protocol [START_REF] Clough | Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[END_REF].

Positive transformants were screened as stated earlier [START_REF] Rathor | A novel protein from Ectocarpus sp. improves salinity and high temperature stress tolerance in Arabidopsis thaliana[END_REF]. Expression of the transgene was analyzed in these lines using quantitative real time polymerase chain reaction (qRT-PCR) as described in later section. For further experiments two homozygous independent transgenic lines named as EsHSP-Ox1 and EsHSP-Ox2 (for 35S promoter) and three homozygous independent lines for stress inducible promoter (EsHSP-A, B, and C) were used.

Plasmid construction for subcellular localization

To generate the transformation vector, pEarleyGate 103 (C-GFP-HIS) [START_REF] Earley | Gateway-compatible vectors for plant functional genomics and proteomics[END_REF]) was obtained from ABRC. Stop codon was removed from the coding sequence of EsHSP70 and the entry clone produced as described in previous section. The entry clone harboring full gene sequence without stop codon was then recombined into pEarleyGate 103 (C-GFP-HIS) using LR Clonase TM II Gateway ® as mentioned in previous section.

Analysis of salinity stress tolerance

Seeds of the wild type and transgenic Arabidopsis lines were germinated and grown vertically on half strength Murashige and Skoog (MS) solid media (Phytotech, USA) for 4 days.

Uniform 4-day-old seedlings were selected and transferred on 1/2 MS solid media plates containing 100 mM NaCl. Plates were placed vertically for 7 days at 22 °C with 16 h light/8 h dark cycle, with light intensity of 100 µmol. photons m -2 s -1 . Primary root length was marked on the day of seedling transfer. After 7 days, plates were scanned using a scanner (Epson Expression 10000 XL, Epson, Ontario, Canada), and increase in root length and the lateral roots number was measured using Image J software (NIH, United StatesUSA). The percent leaf chlorosis was recorded visually on 7 th day after transfer. The seedlings were harvested on the 9 th day to determine the fresh weight, subsequently oven-dried for 5 days at 70 °C for dry weight measurement. All the experiments were conducted in triplicate and 30 plants per each treatment were examined. To investigate if the phenotypes observed in seedlings were evident in plants, seeds of wild type and of EsHSP70-overexpressing lines were stratified for 2 days at 4 °C and were sown directly on Jiffy peat pellets (Jiffy, NB, Canada). 13-day-old plants grown on peat pellets were completely saturated with water to equilibrate amount of water in all peat pellets across the treatments. After three days, they were treated with NaCl at the final concentration of the 100 mM. The peat pellets moisture were kept equally during the experiment by irrigation once every three days. Plants were photographed after 3 weeks and their fresh and dry biomass were measured. Three independent experiments with five replications were performed.

In order to understand the effect of salinity stress on membrane intactness an experiment for electrolyte leakage was performed. Plants were grown and treated as described above.

Following the exposure to salinity stress rosette leaves were harvested from 3 plants in one biological replicate (5 biological replicates) and transferred into vials comprising 20 ml of deionized water. Electrolyte leakage was measured using SympHony SB70C (VWR, ON, Canada) conductivity meter as described by [START_REF] Cao | Modulation of ethylene responses affects plant salt-stress responses[END_REF]).

Plant RNA isolation and quantitative RT-PCR (qRT-PCR)

The expression of abiotic stress associated key marker genes was examined in EsHSP-Ox1 (35S promoter) and EsHSP-A (RD29A promoter) lines. Plants were grown and treated as described above. Total RNA was isolated from leaves at 24 and 120 h after salt stress treatment using GeneJET plant RNA purification kit (Thermoscientific, ON, Canada). After quantification of the RNA using a NanoDrop spectrophotometer (Thermoscientific, ON, Canada), 2 µg of total RNA was treated with RQ1 RNAse free DNAse following the manufacturer's protocol (Promega, ON, Canada) and used for cDNA synthesis using the RevetAID cDNA Synthesis kit according to the manufacturer's instructions (Thermoscientific, ON, Canada). qRT-PCR was conducted on the StepOne plus Real-Time PCR system (Applied Biosystems, Ontario, Canada), using iTaq SYBR green (Bio-RAD, ON, Canada). The relative expression was determined by normalization to the Actin transcript abundance (as an internal control) following the 2 -ΔΔCT method [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF]. Finally, transcript levels were expressed relative to the treatment with lowest expression. The oligonucleotide primer sequences are given in Supplemental Table S1.

Protoplast isolation and transfection

Four weeks old Arabidopsis wild-type (Col-0) plant was used for protoplast isolation and transfection as previously described [START_REF] Yoo | Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[END_REF]). The transformed protoplasts were incubated for 16-18h at room temperature before imaging using an LSM 710 LASER scanning confocal microscope (Carl Zeiss, Canada) following the protocol stated earlier [START_REF] Wu | Tape-Arabidopsis Sandwich -a simpler Arabidopsis protoplast isolation method[END_REF]).

Statistical analyses

Analysis of Variance (ANOVA) was accomplished for all the data followed by Tukey post hoc test at P ≤ 0.05 using Minitab 19.0 software (Minitab LLC, State College, Pennsylvania, USA).

Results

Prediction of transmembrane domain and intracellular localization of EsHSP70

The presence of the transmembrane domains in EsHSP70 (Esi0379_0027) was analyzed using the HMMTOP v.2.0 and TMHMM v.2.0 online prediction servers, however, transmembrane domains was not identified in this protein. SignalP, iPSORT and TargetP 2.0 online servers were employed to analyse the subcellular localization of EsHSP70 protein.

According to the results, the presence of a mitochondrial signal peptide, chloroplast, or thylakoid luminal transfer peptide was ruled out. In plants, it is known that endogenous HSP70 is present in different cellular compartments and it is likely that EsHSP70 in transgenic plants likely to be same. However, final destination in particular cellular compartment may occur by interaction with other proteins. Protein structure prediction was performed based on the PSIPRED method and exhibited the secondary structure of EsHSP70 protein with a MW of 71.9 kDa and an isoelectric point (pI) of 5.0 (Fig. 1 a). The EsHSP70 protein structure was further explored using the SWISS-MODEL server. The best model (77.64% sequence identity) was obtained using bovine Heat shock cognate 71 kDA as a template (SMTL ID: 4fl9.1). This suggested that EsHSP70 is a well-structured protein. Information coming from all these databases indicated that EsHSP70 is a soluble, monomeric protein (Fig. 1 b-d).

Phylogenetic analysis indicated that EsHSP70 considerable diverged from land plants

Phylogenetic analyses of EsHSP70 (Fig. 2) showed the close evolutionary relationships between Ectocarpus sp. sequences and other two brown alga (Saccharina japonica and Undaria pinnatifida). Three sequences from Ectocarpus genus were in the same group as EsHSP70, while these taxa are clustered separately from the Tracheophytes (vascular plants), and Bryophyta (non-vascular land plants). These results implying that these Ectocarpus sp. sequences occurred through duplication and divergent evolution.

EsHSP70 is highly expressed in nucleus

To determine the protein expression pattern, the C-terminal fusion construct of EsHSP70

with GFP tag was transiently expressed in Arabidopsis protoplast. Fluorescence microscopy analysis of protoplast revealed the nucleus localization of the EsHSP70 (Fig. 3). times, respectively). Further up-regulation of this gene in the 35S lines under salinity stress suggests that mRNA is likely more stable under salinity stress [START_REF] Shi | Overexpression of a plasma membrane Na + /H + antiporter gene improves salt tolerance in Arabidopsis thaliana[END_REF].

Expression of EsHSP70 ameliorated salt tolerance in Arabidopsis

To investigate the salt stress tolerance of overexpression lines, 4-day-old seedlings grown on 1/2 MS solid media medium plate were treated with 100 mM NaCl and further grown for 7 days. All the transgenic lines exhibited higher tolerance to salt stress (Fig. 5). After one week following the exposure to salinity stress it was noticed that seedlings of overexpression lines had significantly longer roots (12-13 and 9-10% increase under standard conditions and 35-36 and 30-40% increase under salinity stress for 35S and stress inducible overexpression lines, respectively), higher number of lateral roots per cm of primary root (16-23 and 7-8% decrease under standard conditions and 15-20 and 17-38% increase under salinity stress for 35S and stress inducible overexpression lines, respectively), reduced leaf chlorosis (50-60 and 50-73% for 35S

and stress inducible overexpression lines, respectively), higher fresh weight (10-15 and 10-15% increase under standard conditions and 75-80 and 45-50% increase under salinity stress for 35S

and stress inducible overexpression lines, respectively), and higher dry weight (14-15 and 10-15% increase under standard conditions and 70-80 and 40-42% increase under salinity stress for 35S and stress inducible overexpression lines, respectively) in contrast to the wild type plants (Fig. 5). Under in-vivo condition, wild type plants exposed to the salt stress, displayed severe growth retardation in contrast to transgenic plants which continued to grow better (Fig. 6). In response to salt stress, transgenic plants showed higher fresh weight (50-60 and 40-50% increase under salinity stress for 35S and stress inducible overexpression lines, respectively) and dry weight (60-65 and 45-60% increase under salinity stress for 35S and stress inducible overexpression lines, respectively) in compared to the wild type plants (Fig. 6). Interestingly, no significant differences were observed between transgenic lines generated using 35S promoter and stress inducible promoter under both normal and salt stress condition (Fig. 7).

Expression of EsHSP70 demonstrated reduced leakage of electrolytes

To examine the membrane stability under salt stress condition, 15-day-old wild type and overexpression lines were exposed to 100 mM NaCl. Electrolyte leakage was estimated at 24 and 48 h after salinity stress exposure. Electrolyte leakage at 48 h was significantly lower in both 35S

and stress inducible promoter lines (50-53 and 52-60% in 35S and stress inducible overexpression lines, respectively) in compared to control plants (wild type) suggesting that membranes were highly intact in overexpression lines (Fig. 8). Different letters above the bars represent significant differences according to Tukey's test (p ≤ 0.05). for RAB18 that was 3.9-fold up-regulated in EsHSP-A at 120 h. Contrary to this, EsHSP-Ox1 and EsHSP-A exhibited higher expression level of the stress responsive genes after 24 h and 120 h salt stress exposure (Figs. 9 and 10). The expression level of DREB2A and HSP70 genes was more than 2-fold higher in EsHSP-Ox1 in compared to wild type under both time points of the salt stress condition (Figs. 9a andi, respectively). The expression of these genes was significantly up-regulated in EsHSP-A line at both time points under salinity stress, and the difference was 1.7 and 1.4-fold for DREB2A and 1.7 and 2.4-fold for HSP70 (Figs. 10a andi, respectively). In general, the expression level of RD29A, RD29B, RD26, RD22, and RD20 was enhanced (> 2-fold) at 24 h and reduced at 120 h salt stress conditions (Figs. 9b-f, respectively).

Expression of the stress responsive genes is modulated in

However, the expression of RD29A, and RD26 was significantly different from wild type at 120 h (Figs. 9b andd, respectively). The expression of these genes except RD22 was up-regulated in EsHSP-A line. The difference was 2.0 and 1.8-fold for RD29A, 2.0 and 2.1-fold for RD29B, 1.9 and 2.2-fold for RD26 and 1.9 and 1.5-fold for RD20 (Figs. 10b-f, respectively). The Expression of RD22 was significantly different at 24 h salt stress but not at 120 h in EsHSP-OX1 whereas no significant differences were found in EsHSP-A line at both time points (Figs. 9e and10e). The expression of RAB18 (coding for a protein from the dehydrin family), and LEA14 were also increased by salt stress in both EsHSP-Ox1 and EsHSP-A, at both time points, and this difference was 1.9 and 3.0-fold for RAB18 and 1.6 and 1.9 fold for LEA14 (Figs. 9g andh respectively) in EsHSP-OX1, whereas the difference was 1.5-fold at both time points for RAB18 and 1.5 and 1.3-fold for LEA14 in EsHSP-A line (Figs. 10g and h respectively). Expression of

Discussion

Role of heat shock proteins have been extensively studied in bacteria, plants and animals.

Functions of HSP70 protein in response to abiotic stresses has been investigated in several crop plants including rice, soybean, pepper, and Arabidopsis [START_REF] Lin | Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana[END_REF][START_REF] Jung | Genome-wide expression analysis of HSP70 family genes in rice and identification of a cytosolic HSP70 gene highly induced under heat stress[END_REF][START_REF] Sarkar | Functional analysis of Hsp70 superfamily proteins of rice (Oryza sativa)[END_REF]Zhang et al. 2015a;Zhang et al. 2015b). Transcriptomics analysis of the brown alga Ectocarpus sp. identified HSP70 gene in this organism and similarity searches revealed that this protein is well conserved. This gene was consistently up-regulated in Ectocarpus sp. under different abiotic stresses suggesting that it plays an important role to provide protection against these physiological conditions in this organism [START_REF] Ritter | Transcriptomic and metabolomic analysis of copper stress acclimation in Ectocarpus siliculosus highlights signaling and tolerance mechanisms in brown algae[END_REF]. To understand the function of this gene we expressed it in A. thaliana under the 35S and stress inducible promoter RESPONSIVE TO DESSICATION 29A (RD29A). This is the first report on the overexpression of Ectocarpus sp. HSP70 improving high salinity stress tolerance in land plants.

Heat shock proteins, also known as chaperons, plays critical role in maintaining the metabolic and structural integrity of cells by ensuring proper folding of misfolded or unfolded proteins and preventing protein aggregation under abiotic stress conditions [START_REF] Vierling | The roles of heat shock proteins in plants[END_REF][START_REF] Sung | Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis. Evidence for pleiotropic consequences[END_REF][START_REF] Wang | Role of plant heatshock proteins and molecular chaperones in the abiotic stress response[END_REF]. HSPs are shown to be induced by salinity stress in rice [START_REF] Ngara | Understanding the complex nature of salinity and drought-stress response in cereals using proteomics technologies[END_REF], wheat [START_REF] Sobhanian | Changes in the plant proteome resulting from salt stress: toward the creation of salttolerant crops?[END_REF] and Poplar [START_REF] Manaa | Salt and genotype impact on plant physiology and root proteome variations in tomato[END_REF]. Previously, transgenic plants have been generated to promote abiotic stress tolerance by expressing HSP70 gene from microbes and other land plants. HSP70 from these two types of organism had higher sequence similarity to the HSP70 of other plants. For instance, overexpression of HSP70 from

Erianthus arundinaceus has been shown to enhance salinity and drought stress tolerance of sugarcane [START_REF] Augustine | Erianthus arundinaceus HSP70 (EaHSP70) overexpression increases drought and salinity tolerance in sugarcane (Saccharum spp. hybrid)[END_REF]. This increase in tolerance was due to higher membrane stability and up-regulation of abiotic stress responsive genes including DREB2A, LEA, RD29 and Higher salt stress tolerance was ascribed to the higher expression level of SOS1 and APX1 in transgenic plants. In another study, drought tolerance was enhanced by overexpression of NtHSP70-1 [START_REF] Cho | Over-expression of tobacco NtHSP70-1 contributes to drought-stress tolerance in plants[END_REF]. Similarly, MsHSP70-expressing transgenic Arabidopsis showed higher drought tolerance [START_REF] Li | Molecular cloning and functional analysis of the drought tolerance gene MsHSP70 from alfalfa (Medicago sativa L.)[END_REF]. Its multiple sequence alignment showed that it had higher sequence similarity with other land plants including wheat, rice, corn and peas.

Transgenic Arabidopsis lines expressing chrysanthemum HSP70 exhibited enhanced tolerance to abiotic stress [START_REF] Song | A chrysanthemum heat shock protein confers tolerance to abiotic stress[END_REF]. Taken together, all these studies suggest that HSP70 plays an important role in abiotic stress tolerance in plants. However, HSP70 gene used in these studies had high sequence similarity to the HSP70 of the plants. In the current study, we isolated the HSP70 gene from brown algal model Ectocarpus. sp. Phylogenetic analysis indicated that this gene got considerably diverged from HSP70 in land plants as the close relatives were only two brown algae and other algae from Xanthophyceae family. This gene has no similarity to HSP70 of the plants suggesting that this is a novel HSP70. Expression of EsHSP70 in A. thaliana significantly improved plant tolerance to salinity stress in all lines, irrespective of the promoter used. It is worth mentioning that under both salinity stress and normal conditions, there was no positive correlation between the EsHSP70 transcript abundance and phenotypic data. Although, EsHSP70 expression was higher in 35S lines (constitutive over-expressed lines) than that of RD29A lines (stress-inducible lines), they exhibited a similar phenotype (Figures 5,7,and 8).

This might be due to the similar protein expression in these lines. Indeed, there is no correlation between the transcript level and the final protein expression. It has been shown that several processes such as translation rates, translation rate modulation, delay in protein synthesis, and protein transport are significantly contribute in protein expression. This was further observed in the transcriptomics and proteomics studies [START_REF] Maier | Correlation of mRNA and protein in complex biological samples[END_REF][START_REF] Vogel | Insights into the regulation of protein abundance from proteomic and transcriptomic analyses[END_REF][START_REF] Liu | On the dependency of cellular protein levels on mRNA Abundanceabundance[END_REF]. Furthermore, the elevated expression level of EsHSP70 in constitutive over-expressed lines, might already very high for the translational system leading to a saturation or a plateau level.

Plants face alterations in their growing environment and responds to these variations by adjusting their metabolism and physiology. The crucial changes during these alterations include osmotic adjustment by production and accumulation of compatible solutes to maintain turgor pressure, exclusion of Na + ions, modulation of root architecture, shoot growth, organization of leaves, leaf senescence, flowering time and biomass accumulation [START_REF] Munns | Comparative physiology of salt and water stress[END_REF][START_REF] Sun | Salt modulates gravity signaling pathway to regulate growth direction of primary roots in Arabidopsis[END_REF][START_REF] Park | A rice immunophilin gene, OsFKBP16-3, confers tolerance to environmental stress in Arabidopsis and rice[END_REF]. Phytohormone The phytohormone ABA plays a crucial role in plant tolerance to abiotic stresses [START_REF] Finkelstein | Abscisic acid signaling in seeds and seedlings[END_REF]. ABA is involved in the activation of the various stress responsive genes, however, some of them are activated through an ABA-independent pathway [START_REF] Zhu | Salt and drought stress signal transduction in plants[END_REF][START_REF] Yamaguchi-Shinozaki | Organization of cis-acting regulatory elements in osmotic-and cold-stress-responsive promoters[END_REF]. Stress responsive genes in plant contain two main classes of cis acting elements referred as ABRE and DRE in the promoter regions. ABRE functions in ABA dependent signaling pathway and DRE functions in ABA independent pathway [START_REF] Kim | An ABRE promoter sequence is involved in osmotic stress-responsive expression of the DREB2A gene, which encodes a transcription factor regulating drought-inducible genes in Arabidopsis[END_REF]). In the present work, we analyzed the expression level of various key marker genes associated to abiotic stress tolerance in Arabidopsis (i.e., DREB2A, RD29A, RD29B, RD26, RD22, RD20, RAB18, LEA14, HSP70, SOS1 and NHX1).

A DRE binding transcription factor DREB2A contains ethylene responsive factor elements in promoter region (ERF/APETALA2). It functions as transcriptional activator of downstream stress responsive genes by binding to DRE sequences present in promoter regions [START_REF] Liu | Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperatureresponsive gene expression, respectively, in Arabidopsis[END_REF][START_REF] Nakashima | Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration-and high-salinity-responsive gene expression[END_REF][START_REF] Sakuma | DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydrationand cold-inducible gene expression[END_REF]. The DREB2A expression was significantly up-regulated in EsHSP70-expressing Arabidopsis under salt stress condition (at both 24 and 120 h) when compared to the wild wild-type plant (Figs. 9a and10a). It was shown that the overexpression of the DREB2A activated the expression of several downstream genes including RD29A, RD29B, RD17 and LEA14 (Sakuma et al. 2006a). Likewise, the expression of these genes i.e., RD29A, RD29B and LEA14 was significantly up-regulated in EsHSP70-expressing Arabidopsis when compared to the wild wild-type plant (Figs. 9 and 10b, c, and h, respectively).

Promoter region of these genes contain DRE suggesting that expression of these genes was regulated by DREB2A (Sakuma et al. 2006b). Therefore, higher expression of these genes in transgenic lines can be ascribed to the EsHSP70-mediated activation of the DREB2A. RD26

encodes NAC transcription factor functions in ABA dependent pathway as its expression was up-regulated by salinity, drought, and ABA [START_REF] Fujita | A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway[END_REF]). RD20 encodes a Ca 2+ binding protein functions is directly regulated by RD26 as the expression of this gene was up-regulated in transgenic plants overexpressing RD26 [START_REF] Fujita | A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway[END_REF]). Our result showed the higher expression of RD26 and its target gene RD20 in EsHSP70-expressing Arabidopsis line at both time points for RD26 and at 24 h for RD20 (Figs. 9 and 10d and f, respectively).

Salinity stress causes disorganization of membrane and therefore results more electrolyte leakage [START_REF] Tuteja | Mechanisms of high salinity tolerance in plants[END_REF]. Electrolyte leakage is widely used to understand and measure the plant tolerance to abiotic stresses. It determines the intactness of cellular membranes under abiotic stress condition. In this study, a lower leakage of electrolytes was found in EsHSP70-expressing plants under salt stress condition suggesting higher membrane stability in transgenic lines.

Several LEA proteins accumulated in response to abiotic stresses in plants. These proteins protect macromolecules such as nucleic acid and enzymes by balancing redox and stabilization of cellular membranes [START_REF] Baker | Sequence and characterization of 6 Lea proteins and their genes from cotton[END_REF][START_REF] Ingram | The molecular basis of dehydration tolerance in plants[END_REF][START_REF] Veeranagamallaiah | Group 1 and 2 LEA protein expression correlates with a decrease in water stress induced protein aggregation in horsegram during germination and seedling growth[END_REF][START_REF] Jia | Overexpression of Late Embryogenesis Abundant 14 enhances Arabidopsis salt stress tolerance[END_REF]. Moreover, the expression level of LEA14 in EsHSP70-expressing Arabidopsis was significantly higher than that of control (Figs. 9 and 10h). In support of our result, [START_REF] Jia | Overexpression of Late Embryogenesis Abundant 14 enhances Arabidopsis salt stress tolerance[END_REF] demonstrated that overexpression of LEA14 increased salt stress tolerance [START_REF] Jia | Overexpression of Late Embryogenesis Abundant 14 enhances Arabidopsis salt stress tolerance[END_REF].

The expression of HSP70 was significantly up-regulated (> 2 fold) under salt stress condition in the transgenic line in contrast to WT (Fig. 9 and10i). Similarly, overexpression of HSP70 improved plant tolerance to high salinity, drought and temperature stress in Arabidopsis [START_REF] Wang | Role of plant heatshock proteins and molecular chaperones in the abiotic stress response[END_REF]) . To improve plant salt tolerance, it is important to prevent the entry of Na + into plants to avoid its accumulation in the cytoplasm or organelles, except in the vacuoles. If plant fails to restrict movement of Na + from outside environment then it stores these toxic ion into vacuoles by increased activity of transporters, and utilizes K + and other compatible solutes to balance the osmotic differences in cytoplasm thus reduces the concentration in cytoplasm to prevent damage to cellular machinery [START_REF] Shabala | Learning from halophytes: physiological basis and strategies to improve abiotic stress tolerance in crops[END_REF]. In Arabidopsis, SOS1 has been shown localized at plasma membrane [START_REF] Shi | The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na + /H + antiporter[END_REF][START_REF] Qiu | Regulation of SOS1, a plasma membrane Na + /H + exchanger in Arabidopsis thaliana, by SOS2 and SOS3[END_REF][START_REF] Quintero | Reconstitution in yeast of the Arabidopsis SOS signaling pathway for Na + homeostasis[END_REF][START_REF] Shi | The putative plasma membrane Na + /H + antiporter SOS1 controls long-distance Na + transport in plants[END_REF]. Based on its expression in different tissues it was suggested that it plays diverse role in Na + efflux from roots, reducing concentration of Na + ions in cytoplasm [START_REF] Zhu | Salt and drought stress signal transduction in plants[END_REF].

Overexpression of SOS1 increased tolerance to salt stress [START_REF] Shi | Overexpression of a plasma membrane Na + /H + antiporter gene improves salt tolerance in Arabidopsis thaliana[END_REF]. AtNHX1 encodes Na + /H + antiporter localized on tonoplast membrane of vacuoles. It functions in sequestration of Na + ions into vacuoles [START_REF] Blumwald | Sodium transport and salt tolerance in plants[END_REF]. Overexpression of NHX1 increased salt tolerance in Arabidopsis [START_REF] Gaxiola | Drought-and salt-tolerant plants result from overexpression of the AVP1 H + -pump[END_REF]) and rice [START_REF] Ohta | Introduction of a Na + /H + antiporter gene from Atriplex gmelini confers salt tolerance to rice[END_REF]). In our study, higher expression of the SOS1 and NHX1 was observed in EsHSP70-expressing lines in compared to control under salt stress condition (at 24 and 120 h) (Figs. 9 and 10j and k, respectively). Together, these results suggest that overexpression of EsHSP70 improves salinity stress tolerance by transcriptional activation of Arabidopsis stress responsive genes.

Conclusions

In the present study, heterologous expression of the heat shock protein 70, from the brown alga Ectocarpus sp., increased salt stress tolerance in Arabidopsis. According to the gene expression analysis, higher tolerance of HSP70-expressing Arabidopsis can be ascribed to the higher expression of the stress-responsive genes involved in ABA-induced abiotic stress tolerance, K + homeostasis, and Na + sequestration. Our results suggest that, in addition to the land plants and microbes, brown algae can be employed as a genetic source to generate transgenic plants with promoted tolerance to various abiotic stresses.

Fig 1 .

 1 Fig 1. Structure prediction of EsHSP70 Protein.

Fig. 2 .

 2 Fig. 2. Phylogenetic analysis of HSP70 in different species. The numbers of node indicate the percentages of bootstrapping after 500 replications. Phylogenetic tree was generated using Maximum Likelihood method and JTT matrix-based model. Tracheophytes (vascular plants), Chlorophyta (green algae), Bryophyta (non-vascular land plants), Opalinata (non-phagotrophic heterokonts), Oomycote (fungus), Pelagophyceae (heterokont algae), Bacillariophyta (diatoms), Xanthophyceae (yellow-green algae), Eustigmatophytes eustigmatophytes (eukaryotic algae), and Phaeophyceae (brown algae).

Fig. 3 .

 3 Fig. 3. Subcellular localization of EsHSP70. The coding region of EsHSP70 without stop codon was fused to GFP under control of the constitutive 35S cauliflower mosaic virus promoter (CaMV35S promoter). Arabidopsis protoplast was transformed with p35S::EsHSP70-GFP by using PEG-mediated protocol. Scale bars = 20 µm.

Fig. 4 .

 4 Fig. 4. Expression of EsHSP70 in two independent lines under the 35S promoter and three independent lines harboring the stress inducible (RD29A) promoter under control (normal condition without salt stress) and salt stress conditions. The values on the bars indicate fold change ratio compared to the line with the lowest EsHSP70 expression, i.e. EsHSP-C in normal condition. Different letters above the bars represent significant differences according to Tukey's test (p ≤ 0.05).

Fig. 5 .

 5 Fig. 5. Salt stress tolerance in control and EsHSP70-expressing Arabidopsis under in-vitro condition. (a, g) WT, (b, h) EsHSP-A, (c, i) EsHSP-B, (d, j) EsHSP-C, (e, k) EsHSP-Ox1, (f, l) EsHSP-Ox2. Wild type, 2 independent 35S promoter (EsHSP-Ox1-2,) and 3 independent stress inducible promoter (EsHSPA-C) transgenic Arabidopsis lines were treated with 0 (control) and 100 mM NaCl. (a) to (f), seedlings grown under salt stress conditions (100mM NaCl). (g) to (l), seedlings grown in normal conditions. Seedlings were photographed 9 days after transfer to the control and 100 mM NaCl solid media. (m) root length, (n) number of lateral roots per cm of primary root, (o) fresh weight, (p) dry weight and (q) leaf chlorosis of the wild type and transgenic plant expressing HSP70. Values correspond to the means ± SE (n = 3). Different letters above the bars represent significant differences according to Tukey's test (p ≤ 0.05).

Fig. 6 .

 6 Fig. 6. Salt stress tolerance in wild type and EsHSP70-expressing Arabidopsis under in-vivo condition. Wild type, 2 independent 35S promoter (EsHSP-Ox1-2,) and 3 independent stress inducible promoter (EsHSPA-C) transgenic Arabidopsis lines were grown for 13 days under normal condition and subsequently treated with no (control) and 100 mM NaCl. (a) WT, (b) EsHSP-A, (c) EsHSP-B, (d) EsHSP-C, (e) EsHSP-Ox1, (f) EsHSP-Ox2, (g) fresh weight and (h) dry weight. The plants were photographed after 3 weeks. Values correspond to the means ± SE (n = 3). Different letters above the bars represent significant differences according to Tukey's test (p ≤ 0.05).

Fig. 7 .

 7 Fig. 7. Phenotype of wild type and 2 independent 35S promoter (EsHSP-Ox1-2,) and of 3 independent stress inducible promoter (EsHSPA-C) transgenic Arabidopsis plants, expressing EsHSP70, in normal conditions. (a) WT, (b) EsHSP-A, (c) EsHSP-B, (d) EsHSP-C, (e) EsHSP-Ox1, (f) EsHSP-Ox2.

Fig. 8 .

 8 Fig. 8. Electrolyte leakage in wild type and EsHSP70-expressing Arabidopsis under salt stress condition. Wild type, 2 independent 35S promoter (EsHSP-Ox1-2) and of 3 independent stress inducible promoter (EsHSPA-C) transgenic Arabidopsis plants were grown and treated as described in materials and methodd section. Values correspond to the means ± SE (n = 5).

  mechanism by which EsHSP70 improved salt tolerance in Arabidopsis, the gene expression level of various stress responsive genes including DREB2A (Dehydration-Responsive Element-Binding Protein 2A), RD29A (Response to Desiccation 29A), RD29B (Response to Desiccation 29B), RD26 (Response to Desiccation 26), RD22 (Response to Desiccation 22), RD20 (Response to Desiccation 20), RAB18 (Responsive to ABA), LEA14 (Late Embryogenesis Abundant), HSP70 (Heat Shock Protein 70), SOS1 (Salt Overly Sensitive 1) and NHX1 (Sodium/Hydrogen Exchanger 1) was measured in EsHSP-Ox1(35S promoter) and EsHSP-A (RD29A promoter) by qRT-PCR. No significant change in transcript level of the studied genes was observed between wild type and transgenic lines in normal condition except

Fig 9 .Fig. 10 .

 910 Fig 9. Expression of stress inducible genes in response to salt stress in EsHSP-Ox1(35S promoter) and wild type plants. qRT-PCR results indicating the transcript levels of various stress inducible genes in the wild type and EsHSP70-Arabidopsis under normal (control) and salt stress condition (100 mM NaCl for 24 h and 120 h). Actin was used as the internal control and transcript levels were normalized to the individual with the lowest expression. Values under the bars indicate fold difference. Default font values represent up-regulation while italicised values represent down-regulation. Data represents mean ± SE (n=3). (a) DREB2A, (b) RD29A, (c) RD29B, (d) RD26, (e) RD22, (f) RD20, (g) RAB 18, (h) LEA14, (i) HSP70, (j) SOS1 and (k)

ERD.

  It has been reported that overexpression of HSP70 gene (T34) from Trichoderma harzianum improved salinity stress tolerance in Arabidopsis (Montero-Barrientos et al. 2010).

FundingB

  .P.'s lab was supported by NSERC-DG grant 1177546, MITACS and France-Canada Research Fund (FCRF). P.R. was supported by an NSERC Industrial Postgraduate Scholarship and a MITACS Globalink Research Award. P.P. and T.T. were also supported by the Agence Nationale de la Recherche via the investment expenditure program IDEALG Grant ANR-10-BTBR-04. CRediT authorship contribution statement Pramod Rathor: Conceptualization, methodology, validation, formal analysis, investigation, data curation, writing-original draft preparation, writing-review and editing, visualization. Tudor Borza: Conceptualization, methodology, validation, formal analysis, investigation, data curation, writing-original draft preparation, writing-review and editing, visualization. Ramin Bahmani: formal analysis, investigation, validation, visualization, writingreview & editing. Sophia Stone: writingreview & editing. Thierry Tonon: resources, writing-review and editing, funding acquisition. Svetlana Yurgel: Writingreview & editing. Philippe Potin: resources, writing-review and editing, funding acquisition. Balakrishnan Prithiviraj: Conceptualization, methodology, supervision, project administration, writing-review and editing, funding acquisition, resources.

Data availability

Data are available for the authors upon reasonable request.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal 629 relationships that could have appeared to influence the work reported in this paper.