Plant, Cell and Environment (2016) 39, 62–79

doi: 10.1111/pce.12591

Original Article

SlDREB2, a tomato dehydration-responsive element-binding 2 transcription factor, mediates salt stress tolerance in tomato and Arabidopsis Imène Hichri1*, Yordan Muhovski2, André Clippe3, Eva Žižková4, Petre I. Dobrev4, Vaclav Motyka4 & Stanley Lutts1 1

Groupe de Recherche en Physiologie Végétale (GRPV), Earth and Life Institute – Agronomy (ELI-A), Université catholique de Louvain (UCL), B-1348 Louvain-la-Neuve, Belgium, 2Département Sciences du vivant, Centre wallon de Recherches Agronomiques, B-5030 Gembloux, Belgium, 3Institut des Sciences de la Vie (ISV), Université catholique de Louvain (UCL), B-1348 Louvain-la-Neuve, Belgium and 4Institute of Experimental Botany, Academy of Sciences of the Czech Republic, 165 02 Prague 6, Czech Republic

ABSTRACT

INTRODUCTION

To counter environmental cues, cultivated tomato (Solanum lycopersicum L.) has evolved adaptive mechanisms requiring regulation of downstream genes. The dehydration-responsive element-binding protein 2 (DREB2) transcription factors regulate abiotic stresses responses in plants. Herein, we isolated a novel DREB2-type regulator involved in salinity response, named SlDREB2. Spatio-temporal expression profile together with investigation of its promoter activity indicated that SlDREB2 is expressed during early stages of seedling establishment and in various vegetative and reproductive organs of adult plants. SlDREB2 is up-regulated in roots and young leaves following exposure to NaCl, but is also induced by KCl and drought. Its overexpression in WT Arabidopsis and atdreb2a mutants improved seed germination and plant growth in presence of different osmotica. In tomato, SlDREB2 affected vegetative and reproductive organs development and the intronic sequence present in the 5′ UTR drives its expression. Physiological, biochemical and transcriptomic analyses showed that SlDREB2 enhanced plant tolerance to salinity by improvement of K+/Na+ ratio, and proline and polyamines biosynthesis. Exogenous hormonal treatments (abscisic acid, auxin and cytokinins) and analysis of WT and 35S::SlDREB2 tomatoes hormonal contents highlighted SlDREB2 involvement in abscisic acid biosynthesis/signalling. Altogether, our results provide an overview of SlDREB2 mode of action during early salt stress response.

Salinity is among the most limiting parameters of crop productivity and causes fruit quality deterioration. It creates a water shortage for plants, and presents in addition a toxic component because of excessive sodium and chloride ions accumulation within plant tissues. Early perception and transduction of the salt stress signal in plants are mainly controlled by transcription factors (TFs; Ouyang et al. 2007; Li et al. 2009). DREB (dehydration-responsive elementbinding) regulators belong to the widespread and further subdivided AP2 (APETALA2)/ERF superfamily (Xu et al. 2011). The AP2/ERF family encompasses no less than 147 and 149 members in Arabidopsis (Arabidopsis thaliana) and grapevine (Vitis vinifera L.), respectively, and is also largely represented in rice (Oryza sativa L.) for instance (Nakano et al. 2006; Licausi et al. 2010). This substantial number of AP2/ERF candidates is consistent with their involvement in many aspects of plant lifespan and abiotic stresses response (Licausi et al. 2010). Typical feature of a DREB TF consists in a single repetition of the highly conserved AP2 or ERF domain of ∼60 amino acids, responsible for DNA-specific binding (Sakuma et al. 2002). Based on the conservativeness of the DNAbinding domain, the DREB subgroup is further divided into six clades (A1 to A6) according to a classification reported in Sakuma et al. (2002). In Arabidopsis, the clade A1 comprises DREB1-like regulators and includes six genes, while the clade A2 includes eight DREB2-type (DREB2 A to H) proteins (Sakuma et al. 2006a). However, a new nomenclature proposed by Nakano et al. (2006) and based on gene structure along with conserved motifs outside the AP2 domain indicates that these same DREB2 regulons are enclosed in the subgroup IV (a and b) of the 12 groups forming the Arabidopsis AP2/ERF family. DREB are known to play crucial roles in abiotic stresses response. More specifically, DREB1-type regulators are implicated in low temperatures response, while DREB2 TFs are likely involved in drought, and to a lesser extent in heat and salinity responses, in abscisic acid (ABA) dependent or

Key-words: Arabidopsis thaliana; Solanum lycopersicum; DREB2; salinity tolerance.

Correspondence: S. Lutts. E-mail: [email protected] *Current address: Institut National de la Recherche Agronomique, Institut Sophia Agrobiotech (ISA), UMR INRA 1355, CNRS 7254, Université de Nice-Sophia Antipolis, 400 route des Chappes, BP167, F-06903, Sophia Antipolis Cedex, France. 62

© 2015 John Wiley & Sons Ltd

SlDREB2 mediates salt tolerance in tomato 63 independent pathways (reviewed in Mizoi et al. 2012). However, this classification is not absolute and some DREB2 regulons can participate both to low temperatures, salt and drought responses (Egawa et al. 2006; Chen et al. 2011). Interestingly, cold differently influences accumulation of the wheat (Triticum aestivum L.) WDREB2 transcript forms (α, β and γ) from drought and salt indicating that alternative splicing is another regulatory mechanism of DREB2 TFs (Egawa et al. 2006). Further regulatory events related to DREB2 stability have been reported, since AtDREB2A is in fact degraded by the 26S proteasome under non-stress conditions, through its negative regulation domain (NRD; Sakuma et al. 2006a; Qin et al. 2008; Chen et al. 2011). As a consequence, Arabidopsis plants overexpressing DREB2A truncated protein lacking its NRD showed strong growth retardation, and enhanced tolerance to drought, salinity and heat, consecutive to the induction of many genes related to the acquisition of these stresses tolerance (Sakuma et al. 2006a,b). Although numerous DREB2 regulators have been reported to be induced by salt stress, only few of them have been characterized in planta, and encompass Arabidopsis AtDREB2A, AtDREB2B and AtDREB2C (Nakashima et al. 2000; Lee et al. 2010), soybean (Glycine max) GmDREB2 (Chen et al. 2007), poplar (Populus euphratica) PeDREB2 (Chen et al. 2009), rice (O. sativa L.) OsDREB2A (Cui et al. 2011) and the desert plant Eremosparton songoricum EsDREB2B (Li et al. 2014). Cultivated tomato (Solanum lycopersicum L.) represents a worldwide essential crop, but is a salt-sensitive glycophyte species.TFs involved in salinity response have been hardly characterized so far in tomato and belong to bZIP-type TFs (Orellana et al. 2010) or to the zinc finger family (Hichri et al. 2014). In this study, we isolated and characterized for the first time in tomato a novel regulator belonging to the DREB2 family, named SlDREB2. SlDREB2 expression pattern was examined during plant development and in early stages of seedling establishment by investigation of its promoter activity, as well as in response to osmotic stresses and hormonal treatments. SlDREB2 overexpression was conducted in WT Arabidopsis, atdreb2a insertional mutants and tomato to evaluate plant tolerance to salinity through measurements of physiological and biochemical stress response parameters. Finally, a transcriptome comparison between a SlDREB2 overexpressing line and WT under salinity conditions was performed.

MATERIALS AND METHODS Plant material and osmotic stress/hormonal treatment assays Tomato (S. lycopersicum L. cv Ailsa Craig) seeds were germinated on Whatman 3MM paper soaked with sterile water in a Petri dish, then transferred to trays filled with a perlitevermiculite mix (1:3 v/v) for 10 d, as described in Hichri et al. (2014). Four-week-old plants dedicated to hydroponics were transferred to 52 L tanks containing aerated half-strength Hoagland nutrient solution (Hichri et al. 2014), and grown for additional 2 weeks before stress treatments in a growth © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

chamber at 24 °C/22 °C under a 16 h day/8 h night photoperiode.Arabidopsis (A. thaliana L. cv Columbia) were grown in individual pots filled with soil at 20 °C/19 °C under a 16 h day/8 h night photoperiode at 320 μmol m−2 s−1. Arabidopsis atdreb2A T-DNA insertional mutants were ordered from the Nottingham Arabidopsis Stock Centre (NASC ID: N597569). For in vitro culture and plant transformation, sterilized tomato seeds were germinated on Murashige and Skoog (MS) medium containing or not kanamycin (100 mg L−1) at 24 °C under a 16h day/8h night regime. Arabidopsis seeds were cold-treated for 2 d at 4 °C, then plated on half-strength MS medium supplemented either with kanamycin (35 mg L−1) for transgenic plants (overexpressors) or hygromycin (15 mg L−1) for T-DNA complemented mutants. For salinity (125 mm NaCl) treatments, salt was directly added to the culture tank. For additional stresses including NaCl (150 mm), KCl (150 mm) and drought (air drying) as well as phytohormonal assays, 3-week-old tomato seedlings were grown in smaller tanks with a capacity of 4.5 L. Cytokinin (zeatin; mixed isomers including approximately 80% trans-zeatin, Sigma Z0164), abscisic acid (ABA; Sigma A7383) and auxin (indole-3-acetic acid IAA; Sigma I2886) were used at final concentration of 10 μM. Abiotic stresses and hormonal treatments lasted 3 h, starting on 10:00 a.m. At least three plants were sampled at each time point of analysis.

Analysis of SlDREB2 expression Tomato total RNA was extracted as described in Hichri et al. (2010). After quantification and purity evaluation, an aliquot of 2 μg DNAse-treated RNA was used as template for reverse-transcription (RevertAid™ H Minus First Strand cDNA Synthesis Kit, Fermentas, Vilnius, Lithuania) using oligod(T)18 following the instructions in the manual. Transcript levels of the different genes were measured by qRTPCR using SYBR Green on an iCycleriQ (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in triplicate using 0.2 μm each primer, 5 μL SYBR Green mix (BioRad), and 300 ng of DNAse-treated cDNA in a final volume of 10 μL. Negative controls were included in each run. PCR conditions were as following: initial denaturation at 95 °C for 90 s followed by 40 cycles of 95 °C for 30 s, 60 °C for 1 min. Amplification was followed by melting curve analysis to check the specificity of each reaction. Data were normalized according to the SlGAPDH and SlActin or SlEF1α gene expression levels. Normalized expression of SlDREB2 was calculated using the Gene Expression Analysis for iCycleiQ_ Real Time PCR Detection System software from Bio-Rad with a method derived from the algorithms outlined by Vandesompele et al. (2002).

Plasmid constructions and plant transformation SlDREB2 cDNA was amplified with the Pfu DNA polymerase (Promega, Madison, WI, USA) in a final reaction volume of 50 μL following the manufacturer’s instructions, using as template 300 ng of cDNAs synthesized from total RNA

64 I. Hichri et al. extracted from salt stressed (150 mm NaCl, 2 h of stress) tomato roots of plants grown in vitro. SlDREB2 coding sequence was cloned (Gateway™ technology, Invitrogen, Carlsbad, CA, USA) either into the pK7WG2D or to the pH7WG2D binary vectors (Karimi et al. 2002). Constructs were introduced into the Agrobacterium tumefaciens strain GV3101 for Arabidopsis transformation and in the LBA4404 strain for tomato transformation. For subcellular localization, SlDREB2 coding sequence was introduced in frame into the pYFP-attR vector (Subramanian et al. 2006). Isolation, purification and PEGmediated transformation of tomato protoplasts originating from young leaves of in vitro grown plants have been performed as described in Hichri et al. (2010). YFP alone, YFP– SlDREB2 fusion protein, and chlorophyll fluorescences were visualized using a Zeiss 710 confocal microscope (Carl Zeiss, Jena, Germany). YFP was excited at 514 nm and detected between 530 and 570 nm. Images were analysed with the Zen Software (Zeiss). SlDREB2 promoter sequences were cloned into the pKGWFS7 binary vector (Karimi et al. 2002), and tomato stable transformation was adapted from Ellul et al. (2003). For GUS staining, transformed seeds and seedlings were incubated for 16h at 37 °C in GUS staining buffer (100 mm sodium phosphate pH 7.2, 2 mm X-Gluc (5-bromo-4-chloro3-indolyl-beta-D-glucuronic acid, cyclohexylammonium salt), 10 mm EDTA, 2 mm K4Fe(CN)6, 2 mm K4Fe(CN)6, 0.1% Triton X-100). Arabidopsis plants were transformed by the floral dip method (Clough & Bent 1998). For dreb2a insertional mutants complementation, segregating homozygous plants were identified by PCR using a combination of the LBb1.3, AP2LP and AP2RP primers. All experiments were conducted on homozygous transgenic lines selected in the F3 generation.

Malonyldialdehyde, proline, polyamine, ion and phytohormone extraction and quantification MDA was extracted from Arabidopsis seedlings by the thiobarbituric acid reaction as detailed in Quinet et al. (2012). Proline and free polyamines were extracted and quantified as described in Hichri et al. (2014). Mineral quantification was conducted on the third fully expanded leaf from the bottom of the plant as described in Hichri et al. (2014). Phytohormones were extracted from four leaves stage tomato seedlings grown in vitro on MS medium or from leaf 4 of plants grown in hydroponics under control or salt stress treatment. Hormonal analysis and quantification was performed by HPLC (Ultimate 3000, Sunnyvale CA, Dionex, USA) coupled to hybrid triple quadrupole/linear ion trap mass spectrometer (3200 Q TRAP, Applied Biosystems, Foster City, CA, USA) using a multilevel calibration graph with [2H]-labelled internal standards as described by Djilianov et al. (2013) and detailed in Hichri et al. (2014). All measurements were performed separately on at least three distinct plants for each line and each individual sample

was analysed in triplicates, except hormone analysis that were performed in duplicate.

Physiological parameters measurement The stomatal conductance (gs) was measured using an AP4 system (Delta-T Devices; Cambridge, UK) on leaf between 2:00 p.m. and 4:00 p.m. Osmotic potential was measured on leaf 5 as described in Hichri et al. (2014). All measurements were performed in triplicate.

Microarray analysis A commercial Affymetrix (Affymetrix, Santa Clara, CA, USA) tomato microarray representing 43 803 tomato probes was used. Transcripts represented on the array have been selected from RefSeq, UniGene, TIGR Plant Transcript Assemblies and TIGR Gene Indices databases. Total RNA was extracted from transgenic and WT plants grown in hydroponics and submitted to salt (125 mm NaCl) during 48 h. RNA purity and yield were first assessed as described above, and RNA (500 ng) quality again examined with the Agilent 6000 RNA Nano kit by the Agilent Bioanalyzer (Agilent Technologies). Ten μg of total RNA were used for each fluorescence labelling reaction (Invitrogen, Paisley, UK). RNA fluorescence labelling, hybridization, data extraction and gene expression analysis were conducted as described in Quinet et al. (2012). Statistical selection of differentially expressed genes between WT and D4 transgenic tomato lines was based on a minimal 2.5 log2 fold change, together with a P-value ≤ 0.05 for the t-test, for each technical repetition. Annotation of genes was completed by Basic Local Alignment Search Tool (BLAST) searches in the National Center for Biotechnology Information (NCBI) database. Data were submitted to the GEO repository under GSE64010 reference. Primers used for qRT-PCR analyses of candidate genes are provided in Supporting Information Table S2.

Statistical analyses Normality of the data was estimated with Shapiro–Wilk tests and homoscedasticity was verified using Levene’s test. Data were transformed when required to ensure normal distribution. Statistical analyses were conducted with SAS software (SAS System for Windows version 9.1, SAS Institute Inc., Carry, NC, USA). An analysis of variance (anova) using the mean discrimination was performed on all data set using the Student–Newman–Keuls test at the 5% level.

Accession numbers Sequence data can be found in the GenBank data library under the following accession numbers: SlDREB2 (ADZ15315), SlDREB1 (AAN77051), AdERF3 (ADJ67432), SbDREB2A (ADE35085), AtDREB2C (Q8LFR2), GmDREB2A;1 (AFU35562), GmDREB2A;2 (AFU35563), CkDREB2 (AGI78251), PeDREB2L © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

SlDREB2 mediates salt tolerance in tomato 65

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PiSlDREB2::GUS

PpSlDREB2::GUS

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SlDREB2-YFP

YFP

Figure 1. Identification of SlDREB2. (a) Phylogenetic relationships between SlDREB2, GmDREB2A;1, GmDREB2A;2, CkDREB2, AtDREB2C, PeDREB2L, SbDREB2A, CrORCA2, SlDREB1, AdERF3 and OsDREB2A. The phylogenetic tree was constructed according to the neighbour-joining method, using MEGA6 (Tamura et al. 2013). The percentage of reliability of each branch point of the rooted tree, as assessed by the analysis of 1000 trees (bootstrap replicates), is shown on the branch stem. (b) Subcellular localization of YFP and YFP–SlDREB2 in tomato leaf protoplasts (YFP control fluorescence and bright field/ YFP fluorescence). Scale bar indicates 5 μm. (c–d) Histochemical localization of PiSlDREB2 and PpSlDREB2::GUS promoters-driven GUS expression in tomato germinating seedlings grown on control MS medium (c) or on 125 mm NaCl (d). Scale bar indicates 1 mm. Pictures were taken from 3 d post-seeds imbibition until 12 d later.

(ABV03750), CrORCA2 (CAB93939), OsDREB2A (Q0JQF7), ABA-deficient 4 (AK320933), DREB2A (AK325517), ERF109 (EG553122), HKT1 (AK324676), KAN2 (AI491005), MIP (AK247308), MYB48 (AK322126), Na exch (AK320589), PS1 (DQ335097), ABAhydroxy (AW217177), NRT2 (AF092655), SAP11 (AK327173), WRKY3 (BM412776), WRKY40 (AK325041), ZAT11 (EG553756), GAPDH (XM_010322252), Actin (XM_ 004236699) and EF1α (X14449).

RESULTS SlDREB2 encodes a DREB2-type regulator A previous study (Ouyang et al. 2007) allowed the identification of multiple putative TFs induced by salinity in cultivated tomato. Among these candidates, an AP2 encoding gene (initial accession DY523801) was selected for further functional characterization, and named SlDREB2 (GenBank accession number HQ698902). SlDREB2 coding sequence is © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

1371-bp in length, and includes a 1227-bp open reading frame predicted to encode a 408 amino acids (aa) polypeptide of 45.7 kDa. Additionally, cloning of SlDREB2 genomic DNA sequence revealed the presence of a 927 bp intron located into SlDREB2 5’untranslated (UTR) region. SlDREB2 exhibits a highly conserved AP2/ERF domain including Val (position 95) and Glu (position 100) in the β-2 sheet region, which discriminate between the DREB and ERF TFs (Supporting Information Fig. S1). The AP2 domain is directly followed by a serine rich region, considered to putatively drive proteolysis (Sakuma et al. 2006a). Outside the AP2 domain, SlDREB2 and its orthologs SlDREB1, SbDREB2A and AtDREB2C, harbour a conserved N-terminal end and a short stretch of 11 aa rich in acidic residues (SlDREB2 residues 284 to 294; Supporting Information Fig. S1). A phylogenetic analysis including SlDREB2 and 10 additional orthologs (Fig. 1a) first illustrated the similarity of SlDREB2 to SlDREB1, uncharacterized so far. SlDREB2 was also closely related to DREB2 TFs highly induced by osmotic stresses. Indeed, AtDREB2C (Lee et al. 2010),

66 I. Hichri et al. Caragana intermedia DREB2C (Zhu et al. 2013), soybean DREB2A;1 and DREB2A;2 (Mizoi et al. 2013), poplar PeDREB2L (Chen et al. 2011), the extreme halophyte Salicornia brachiata SbDREB2A (Gupta et al. 2010) and rice (O. sativa) OsDREB2A (Matsukura et al. 2010) are differently affected by salinity, drought, and ABA. The basic residues motif RKKK existent (position 18) in SlDREB2 protein represents a putative nuclear targeting signal (NLS; Supporting Information Fig. S1). To verify its subcellular targeting, SlDREB2 was fused in its N-terminal extremity to the yellow fluorescent protein (YFP), and the construct was used to transiently transform tomato protoplasts (Fig. 1b). In accordance to the predicted function of TFs, the YFP-SlDREB2 fusion protein was mainly located in the nucleus, but also in the cytosol (Fig. 1b). Similarly, the free YFP protein was detected both in cytosol and nucleus, as a result of passive diffusion (Fig. 1b).

SlDREB2 is expressed during germination and in vegetative and reproductive organs To investigate SlDREB2 promoter activity during tomato seedling development, two genomic DNA fragments containing either the proximal promoter (PpSlDREB2, 1054 bp) or the proximal promoter including a sequence of the 5′ UTR comprising the 927 bp intron (PiSlDREB2, 2067 bp) were fused to the uidA reporter gene encoding a β-glucuronidase (GUS) and the constructs used for tomato stable transformation. GUS staining of transformed tomato seedlings indicated that PiSlDREB2 activity was mainly detected in the root tip from the very early stages of germination and until radicle full emergence, in the vascular cylinder, as well as at the hypocotyl and cotyledons basis (Fig. 1c). In tomato seedlings, PiSlDREB2 was highly active in the hypocotyl, while a weak GUS activity was visible in the cotyledons (Fig. 1c). PpSlDREB2 showed a reduced GUS activity in the radicle and the main root, and a different localization of GUS staining in the cotyledons (Fig. 1c). Effects of salt on SlDREB2 expression were subsequently investigated on seedlings grown for 5 d in vitro on medium containing 125 mm NaCl. Salt reduced GUS staining in the hypocotyl of PiSlDREB2::GUS plants, while it increased staining in the cotyledons (Fig. 1d). In contrast, salt had no obvious effects on PpSlDREB2 activity. Together, these results indicate that the intronic sequence present in the 5′ UTR of SlDREB2 drives its expression during tomato seedling establishment and in response to salinity. SlDREB2 spatio-temporal expression pattern in vegetative and reproductive organs was then determined by quantitative RT-PCR (qRT-PCR) in adult tomato plants grown in the greenhouse. SlDREB2 was expressed in all vegetative organs tested including roots, stem, young leaves (YL) and old leaves (OL), with a faint preference in roots (Fig. 2a). In generative organs, SlDREB2 transcripts were highly abundant in flowers and buds (Fig. 2b). During tomato fruit development, SlDREB2 was very weakly expressed at the green stage, before a resumption of its expression during tomato ripening and a peak in the red mature fruit (Fig. 2b).

SlDREB2 expression is induced by osmotic stresses SlDREB2 expression pattern during a short-term salt stress (150 mm NaCl, 24 h) was determined by qRT-PCR. In roots (Fig. 2c), accumulation of SlDREB2 transcripts started within one hour after stress initiation, and SlDREB2 expression then gradually increased with time. In YL, SlDREB2 expression progressively increased, peaking 12 h after salinity treatment onset (Fig. 2d). In OL, SlDREB2 was rapidly induced by salt as after 1 h of stress, the amount of transcripts was doubled (Fig. 2d). However, this augmentation was transient, and was followed by a second peak of expression at 8 h (Fig. 2d). In OL, SlDREB2 expression was back to its initial value 12 h after commencement of the stress. Effects of additional osmotic stresses on SlDREB2 expression in tomato roots were investigated by qRT-PCR. As shown in Fig. 2e, short-term (3 h) treatments of KCl (150 mm) and drought (air-drying) enhanced SlDREB2 expression, nevertheless to a lesser extent than NaCl (150 mm).Together, these results pinpointed that SlDREB2 is induced by multiple osmotic stresses, but to a greater extent by salinity.

SlDREB2 improves osmotic stress tolerance in Arabidopsis and complements atdreb2a insertional mutants SlDREB2 was constitutively expressed in Arabidopsis under control of the cauliflower mosaic virus (CaMV) 35S promoter, and analyses were conducted on three F3 homozygous lines (L4, L5 and L11) showing the highest germination capacity under salt stress (Fig. 3, Supporting Information Fig. S2). Effects of SlDREB2 overexpression on Arabidopsis seeds germination on control medium (Supporting Information Fig. S3) or in presence of NaCl (125 mm), KCl (150 mm) and mannitol (300 mm) were examined 48 h after sowing (Fig. 3a). While no differences between WT and transgenic lines were detected on control medium (Supporting Information Fig. S3), all transgenic lines showed significantly higher germination percentage than WT on the stressing media (Fig. 3a). Similarly, primary root length was estimated 7 d after germination (Fig. 3b). While under control conditions, transgenic and WT lines had a similar growth (between 2.8 and 3.4 cm), addition of salt had a significantly greater inhibitory effect on WT roots (0.4 cm) than on L4 (1.1 cm), L5 (1.1 cm) and L11 (0.8 cm). The effects of salt stress on plant oxidative status was subsequently assessed by measurements of proline and malonyldialdehyde (MDA) concentrations. Analyses were conducted on Arabidopsis seedlings grown two weeks in vitro either on control medium or on medium containing 125 mm NaCl. Under control conditions, 35S::SlDREB2 and WT lines showed proline concentration ranging between 6 and 12 μmol g−1 FW (Fig. 3c). In the presence of salt, proline contents increased in all types of plants and were significantly higher in L4, L5 and L11 lines than in WT, reaching 87.2, 113.2, 64.2 and 50.7 μmol g−1 FW, respectively (Fig. 3c). MDA © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

SlDREB2 mediates salt tolerance in tomato 67 (a)

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Figure 2. SlDREB2 expression profile during plant development and in response to osmotic stresses. (a–b) qRT-PCR analysis of SlDREB2 spatio-temporal expression profile during tomato plant development, in (a) vegetative organs and (b) reproductive organs. (c–e) QRT-PCR analysis of SlDREB2 expression in response to osmotic stresses: salinity (150 mm NaCl) in (c) roots and (d) young and old leaves. (e) Effects of NaCl (150 mm), KCl (150 mm) or drought (air drying) on SlDREB2 expression in roots (3 h treatment). Actin and GAPDH were used as internal controls. Data represent means and SEs of six replicates, YL, young leaves; OL, old leaves; Ro, roots; St, stem; Flo, flower; Gre, green stage; Bre, breaker stage; Red, red stage of tomato development. Letters indicate values that significantly differ between treatments according to Student-Newman-Keuls test at P-value < 0.05. © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

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Figure 3. Effects of SlDREB2 overexpression in Arabidopsis on tolerance to different abiotic stresses. (a) Germination percentage of 35S::SlDREB2 (L4, L5 and L11) and WT lines on NaCl (125 mm), KCl (150 mm) and mannitol (300 mm). (b) Primary root length of transgenic and WT lines on control or NaCl (125 mm) supplemented media. (c) Proline and malonyldialdehyde accumulation in WT and 35S::SlDREB2 lines grown for 14 d on control or salt (125 mm NaCl) supplemented media. (d) Germination percentage of WT, atdreb2a, vector (V) and SlDREB2-complemented lines on control medium or media supplemented with 50 mm NaCl or 125 mm NaCl. Data represent means and SEs of three replicates (each replicate consisting in a group of 50 plants). Letters indicate values that significantly differ between SlDREB2-transgenic Arabidopsis and control lines according to Student–Newman–Keuls test at P-value < 0.05.

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

SlDREB2 mediates salt tolerance in tomato 69 contents were measured on the same plants. On control medium, transgenic and WT lines showed similar MDA contents (around 7 nmol g−1 FW). Salinity significantly increased MDA contents in WT, but had no effects on transgenic lines. To further address SlDREB2 function, we searched for Arabidopsis lines inactivated for DREB2 expression. Phylogenetic analysis indicated that AtDREB2C was Arabidopsis closest ortholog to SlDREB2 (Fig. 1a). However, since no atdreb2c insertional mutant line was available to our knowledge, we complemented atdreb2a, as AtDREB2A is also induced by salt and drought (Liu et al. 1998). We first raised homozygous atdreb2a insertion line among the segregating population, before complementation with SlDREB2. Four F3 homozygous independent lines L1, L2, L5 and L12 were selected for examination of their germination capacity in presence of different osmotica, 48 h after sowing (Fig. 3d). WT lines, atdreb2a mutants, in addition to atdreb2a plants complemented with empty vector (V) were used as control lines. On control medium, atdreb2a and V lines showed a significantly lower germination percentage (less than 30%) compared with L1, L2, L5 and L12 (more than 80%) or WT (63%). This delay in germination and early seedling establishment was still visible 4 d later, while SlDREB2 complemented lines showed a similar development as WT (Supporting Information Fig. S4). In presence of 50 mm NaCl, all SlDREB2 complemented lines showed significantly higher germination percentage (more than 65%) than WT (32%) and atdreb2a or V (less than 12%) lines (Fig. 3e). On 125 mm NaCl, only L2 displayed a higher germination percentage than all the lines. Overall, these results demonstrate that AtDREB2A is involved in germination and early seedling establishment, and that SlDREB2 is able to restore a WT phenotype in atdreb2a insertional mutants. In addition, SlDREB2 complemented plants are more tolerant to salinity than KO or WT plants, underlining its dominant role in osmotic stresses response.

SlDREB2 mediates ABA signalling in tomato SlDREB2 was then constitutively expressed under CaMV 35S promoter in tomato. Three F2 generation lines, D1, D4 and D5 (Fig. 4; Supporting Information Fig. S2), were selected for further analysis. Transgenic plants displayed several phenotypic differences in comparison with WT plants, specially the flowers at blossom and the sepals (Fig. 4a).Very few flowers were able to overcome the fruit set step, and as a result, only one to two fruits were produced per 35S::SlDRE2 plant (Fig. 4a). Compared with WT, SlDREB2 overexpressors also presented curved leaves, with some visible chlorosis on adult leaves and punctual defects of formation (Fig. 4a). However, no difference in chlorophyll contents between WT and transgenic young leaves were detected on young leaves (Supporting Information Fig. S5). Overall, the 35S::SlDREB2 tomato plants were in general shorter than WT (Fig. 4a). The altered phenotype of SlDREB2 overexpressors suggested an imbalance in hormonal metabolism, and it prompted us to examine in which pathways could SlDREB2 © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

be involved. ABA, auxin (indole-3-acetic acid, IAA) and cytokinins (CKs, zeatin) were exogenously applied on WT tomato roots and SlDREB2 expression was determined by qRT-PCR (3 h treatment). As observed in Fig. 4b, only ABA could significantly (P-value < 0.05) up-regulates SlDREB2 expression.Total phytohormonal contents were subsequently analysed in WT and transgenic tomato seedlings grown in vitro, thus including roots and shoot. As observed in Fig. 4c, transgenic plants tended to accumulate more ABA (significant for D5) and salicylic acid (SA) than WT, while opposite results were obtained for trans-zeatin riboside (tZR), a bioactive and storage form of CK. Expression of few candidate genes differently expressed between WT and transgenic plants grown under control conditions was examined by qRT-PCR. These genes were selected based on a microarray analysis involving the WT and the D4 transgenic lines grown under control conditions (Hichri et al., unpublished results). Expression of a nitrate transporter NRT2;1 and an abscisic acid 8′-hydroxylase 1-like (ABAhydroxy) encoding genes were down-regulated in 35S::SlDREB2 plants in comparison with WT (Fig. 4d). SlWRKY3 and SlWRKY40 commonly induced by salinity (Ouyang et al. 2007) were down-regulated in SlDREB2 overexpressors as well, together with ZAT11 and SAP11. The ZAT family of zinc finger TFs and the Stress Associated Protein (SAP) family members are involved in osmotic stresses response (Sakamoto et al. 2004; Solanke et al. 2009).

Overexpression of SlDREB2 in tomato promoted salinity tolerance Since SlDREB2 responded to multiple abiotic stresses in tomato (Fig. 2), we investigated the effects of salinity (125 mm NaCl) on transgenic and WT tomatoes cultivated in hydroponics (Fig. 5). Plants were cultivated in growth chamber under controlled parameters (Fig. 5a, left panel) or in the greenhouse (right panel). As observed on Fig. 5a, WT plants died as early as 3 d post-stress induction in the greenhouse. In an attempt to decipher and connect both transcriptional and physiological establishment of the tomato salt stress response, we decided to specifically focus on a stress period of 5 d in fully controlled growth chambers. SlDREB2 overexpressors displayed a delayed salt-induced senescence compared with WT plants (Fig. 5a). While chlorosis was visible for all types of plants 10 d after stress initiation, wilting followed by leaf necrosis was more severe in WT plants than in transgenics (Fig. 5a). Effects of salinity on leaf stomatal conductance (gs) and osmotic potential were examined during 10 d after salt stress initiation on leaf 3 and leaf 5, respectively (Fig. 5b; Supporting Information Fig. S6). Under control conditions, D1 had a significantly lower gs than all remaining lines (P-value < 0.05). Salinity sharply decreased gs for transgenic lines and WT, and 5 d after stress initiation this value was ranging between 55 and 70 mmol m−2 s−1 for transgenics, while it dropped to 25 mmol m−2 s−1 for WT (P-value < 0.05). After 10 d, gs value decreased for D4 and was constant for the remaining lines (Fig. 5b). Leaf osmotic potential was significantly lower in WT (−0.89 MPa) than in

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Figure 4. SlDREB2 overexpression affects different aspects of tomato plant development. (a) Phenotype of WT and 35S::SlDREB2 detailed organs or whole tomato plants (8 to 12 weeks old). (b) qRT-PCR analysis of SlDREB2 expression in response to different hormonal treatments (3 h treatment). EF1α and GAPDH were used as internal controls. (c) Phytohormone contents in D1, D4, D5 SlDREB2 overexpressors and WT four leaves tomato seedlings grown in vitro on regular MS medium. (d) qRT-PCR analysis of candidate genes differentially expressed between SlDREB2 overexpressors and WT plants cultivated in hydroponics. Actin and GAPDH were used as internal controls. Data represent means and SEs of three (qRT-PCR) or two (hormones) replicates. Letters indicate values that significantly differ between treatments or between SlDREB2-transgenic tomatoes and control lines according to Student–Newman–Keuls test at P-value < 0.05.

D1 (−0.79 MPa), D4 (−0.76 MPa) and D5 (−0.75 MPa) transgenic plants under control conditions (Fig. 5b). Following salt stress, leaf osmotic potential decreased for all transformants and WT as early as 2 d after treatment onset, being significantly smaller for WT and D1 than for D4 or D5. The osmotic potential subsequently decreased for all tomato plants except D5 until the end of the experiment. Salinity tolerance depends among others on plant capacity to exclude or to compartmentalize sodium (Na+) and to retain potassium (K+). Contents of Na+, K+ (Leaf 3), proline

and polyamines were evaluated in transformants and WT. Before stress initiation, WT, D1 and D4 tomatoes accumulated less than 0.6 g kg−1 dry weight (DW) Na+, while this value was significantly higher in D5 (1.28 g kg−1 DW) (Fig. 6a). Two days after stress onset, sodium contents progressively increased in all plants, and Na+ contents tended to be higher in WT (26.5 g kg−1 DW) than in D1 (23 g kg−1 DW) or D4 (21.1 g kg−1 DW) and D5 (17.7 g kg−1 DW). Similarly, on day 5, D1, D4 and D5 tended to accumulate reduced Na+ contents in comparison with WT. Such a trend was © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

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Figure 5. SlDREB2 overexpression enhances tomato salt tolerance. (a) Effects of salinity treatment (125 mm NaCl) on transgenic and WT plants grown in hydroponics in the growth chamber for 10 d (left panel) or in the greenhouse for 3 d (right panel). (b) Characterization of SlDREB2 transgenic and WT tomatoes physiological parameters: Leaf stomatal conductance (gs) and osmotic potential. Data represent means and SE of three replicates. Letters indicate values that significantly differ between SlDREB2 transgenic tomatoes and WT according to the Student-Newman-Keuls test at P-value < 0.05.

consistently observed during three independent experiments. The difference with WT was statistically significant after 8 d of treatment for D4 and D5 (Supporting Information Fig. S6). Comparable analyses were conducted for K+ determination. Under control conditions, D1 (59.5 g kg−1 DW), D4 (57.3 g kg−1 DW) and D5 (69.1 g kg−1 DW) accumulated a similar K+ concentration to that of WT (58.5 g kg−1 DW). Salinity progressively decreased K+ contents in transgenic and WT plants. After 5 d of salinity treatment, all transgenic lines significantly accumulated more K+ (∼30 g kg−1 DW) than WT (20.4 g kg−1 DW). Taken together, these results emphasize the retention of higher K+ contents (in parallel with reduced Na+ contents) in transgenic tomatoes than in WT over the stress period. Proline and free polyamines (PAs) were extracted from whole tomato plants. Since these compounds may directly contribute to osmotic adjustment, they were expressed on a fresh weight basis. As observed in Fig. 6b, all transgenic lines significantly accumulated up to 50% less proline than WT (3.21 μmol g−1 FW) under control conditions. Salinity increased proline accumulation both in WT and transgenic © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

lines 5 d after stress onset. However, this augmentation was significantly lower for D1 (14.2 μmol g−1 FW), D4 (11.8 μmol g−1 FW) and D5 (14.1 μmol g−1 FW) than for WT (19.7 μmol g−1 FW). PAs such as spermine are anti-stress and anti-senescence molecules permitting plants to counteract abiotic stresses effects (Gill & Tuteja 2010). Under control conditions, transgenic and WT plants accumulated comparable contents of spermine (∼100 to 130 nmol g−1 FW; Fig. 6c). Salinity treatment reduced spermine accumulation both in WT and overexpressors, although this reduction was significant (P-value < 0.05) only for transgenic lines (∼70 nmol g−1 FW). Together, these results demonstrate that under salt stress D1, D4 and D5 accumulate less stressrelated molecules than WT. Finally, total phytohormones were extracted from leaf 4 under control conditions or 5 d after stress initiation (Fig. 6d). Before salinity treatment, D1, D4 and D5 accumulated up to 60% less ABA than WT (1840 pmol g−1 DW). Salt surprisingly decreased ABA accumulation in WT to 50% of its initial value, whereas in contrast, increased ABA in all transgenic lines. Very similar results were observed for

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(c)

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Figure 6. Stress-related molecules accumulating in 35S::SlDREB2 and WT tomatoes exposed to salt (125 mm NaCl) during 5 d. (a) Sodium and potassium accumulation in leaf 3. (b) Proline and (c) spermine contents in transgenic and WT tomato plants. (d) Phytohormonal contents in leaf 4 of transgenic and WT plants. Data represent means and SEs of two (hormones) or three replicates. Letters indicate values that significantly differ between SlDREB2 transgenic tomatoes and WT lines according to Student-Newman-Keuls test at P-value < 0.05.

© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

SlDREB2 mediates salt tolerance in tomato jasmonic acid (JA), while contrasting results were described for SA. Indeed, salinity increased SA more than three times in WT, while it decreased SA in D1 and D4 to half of its initial value (Fig. 6d). Finally, the slight dwarfism of 35S::SlDREB2 plants (Fig. 4a) prompted us to examine gibberellins contents in transgenic and WT plants (Supporting Information Table S1). Physiologically active gibberellins GA1 (in addition to its precursor GA20) and GA4 were less abundant in transgenic tomatoes than in WT, and reverse trends were observed for the catabolic form GA8, as well as for GA19 and GA29. Salinity differently affected accumulation of these gibberellins in WT and transformants (Supporting Information Table S1). Altogether, these results indicate that salinity differently affects phytohormones accumulation in WT and overexpressors, and transgenic plants particularly accumulate more ABA and JA than WT over the stress period.

SlDREB2 regulates multiple physiological and biochemical pathways in tomato A transcriptomic analysis was carried out to identify differentially expressed genes between the D4 transgenic line and WT tomatoes exposed to salinity (125 mm NaCl) during 48 h. A technical repetition was conducted and allowed identification of 603 differentially expressed genes (minimal 2.5 log2 fold change and P-value ≤ 0.05), with 314 and 289 up- and down-regulated genes, respectively. Functional classification of putative gene products indicated that overexpression of SlDREB2 affected multiple processes including transcription, signal transduction, primary and secondary metabolites biosynthesis, phytohormones biosynthesis, photosynthesis and stress response, to name a few (Fig. 7a). Among the differentially expressed genes, 40 were encoding TFs (Table 1). Induced TFs comprised SlDREB2 and a DREB2A-like TF, SlZF2, which is involved in salt-stress tolerance (Hichri et al. 2014), and a probable salt-tolerance B-box type zinc finger protein. The repressed genes were mainly related to regulation of the flowering and circadian rhythm event, photomorphogenesis and jasmonate signalling pathway (Table 1). A high proportion of up-regulated genes in transgenic plants were associated with abiotic stresses response, and encompassed different types of transporters, in addition to secondary metabolites biosynthesis and detoxification enzymes, as well as proteins preserving the polypeptides structure (Table 1). A particular interest was furthermore paid to genes encoding enzymes of the hormonal biosynthesis/signalling pathways. Among up-regulated genes, several were related to ABA metabolism including a phytoene synthase (PS1), an ABA receptor PYL9-like, and the ABA-deficient 4 (ABA4) protein, a putative neoxanthin synthase (Table 1). To validate our microarray data, nine genes were picked up for supplementary qRT-PCR analysis and the corresponding transcripts were measured in WT and in D1, D4 and D5 (Fig. 7b; Supporting Information Table S2), subjected to 48 h of salinity (125 mm NaCl). The selected genes comprised TFs DREB2A, KAN2, ERF109 and MYB48, transporters such as © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

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the sodium/calcium exchanger (NCX), the high-affinity potassium sodium transporter HKT1-like (HKT1) and the MIP aquaporin, and ABA biosynthesis-related genes such as ABA4 and PS1. For all the tested genes, qRT-PCR analysis confirmed the transcriptomic data.

DISCUSSION Despite advances in recent years in understanding DREB2 TFs mode of action in response to salt stress, there were no studies so far simultaneously gathering transcriptional, hormonal, physiological and biochemical data. Based on a microarray study, we identified and characterized SlDREB2 both in tomato and Arabidopsis.

SlDREB2 encodes a DREB2 regulator in tomato DREB2 subgroup includes eight members in Arabidopsis, in which only DREB2 A, B and C (subgroup IVa) have been characterized (Nakano et al. 2006). Heterologous expression of SlDREB2 in Arabidopsis did not lead to any remarkable phenotype. However, atdreb2a mutants showed growth retardation at germination and during early stages of seedling development (Fig. 3), indicating that AtDREB2A takes part in seedling establishment. Investigation of SlDREB2 promoter activity similarly showed that SlDREB2 may interfere with tomato germination and seedling development at very precocious stages (Fig. 1c). Arabidopsis plants overexpressing AtDREB2A (Sakuma et al. 2006a), AtDREB2C (Lee et al. 2010), rice OsDREB2B (Matsukura et al. 2010), poplar PeDREB2L (Chen et al. 2011) or maize ZmDREB2A (Qin et al. 2007) showed dwarfism, highlighting the effects of DREB2 TFs on plant general growth and metabolism. In this regard, SlDREB2 constitutive expression in tomato did also lead to a certain dwarfism (Fig. 4a), supported by reduction of physiologically active GAs contents in transgenic plants (Supporting Information Table S1). Comparable results were reported following SlDREB overexpression in tomato, which resulted in internodes restriction as a consequence of reduced endogenous GAs contents (Li et al. 2012). Overall, the general plant metabolism was modified in transgenic tomato plants under stress conditions in our experiments (Fig. 7). Microarray and candidate genes expression analyses of Arabidopsis plants overexpressing ZmDREB2A-S or AtDREB2C revealed that many stress-inducible genes were up-regulated in transgenic plants even under non-stressed control conditions (Qin et al. 2007; Lee et al. 2010), which may be at the origin of the general growth retardation. SlDREB2 was localized both in nucleus and cytoplasm, suggesting that it may interact with partner(s) in cytosol, or may undergo post-translational modifications or degradation. Indeed, AtDREB2A seems rather inactive and/or unstable in absence of stresses (Sakuma et al. 2006a,b), and is in fact targeted to the 26S proteasome under non-stress conditions following interaction with the DREB2A-Interacting Protein 1 (DRIP1) ubiquitin E3 ligase (Qin et al. 2008; Chen et al. 2011). Additional regulators of DREB2A activity are

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Figure 7. Differentially expressed genes between D4 and WT lines exposed to 48 h salinity (125 mm NaCl). (a) Microarray analysis indicating number and categories of up- and down-regulated genes in tomato D4 transgenic line compared to WT. (b) qRT-PCR analysis of SlDREB2 putative target genes expression in transgenic tomato lines D1, D4 and D5, comparatively to WT tomatoes, exposed to salt stress (125 mm NaCl, 48 h). Actin and GAPDH were used as internal controls. Data represent means and SEs of minimum three replicates. © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

SlDREB2 mediates salt tolerance in tomato

Accession

Description

Log2 fold

Up-regulated transcription factors TA52862_4081 SlDREB2 AK319264 Protein LHY-like AK329161 B-box type zinc finger family protein salt tolerance BF096555 Transcription factor bHLH80-like isoform 1 AK322126 MYB48 AI491005 Probable transcription factor KAN2-like AK325517 DREB2A-like BT013336 SlZF2 Down-regulated transcription factors AK326408 Probable WRKY transcription factor 40-like AW624869 Zinc finger protein CONSTANS-LIKE 9-like EU670750 SlNAC1 TA47600_4081 GIGANTEA-like AK327683 Jasmonate ZIM-domain protein 1 EG553122 Ethylene-responsive transcription factor ERF109-like AK319263 Scarecrow-like transcription factor PAT1-like isoform 1 EF091574 SlTCP3 BP879017 Transcription factor MYC2-like isoform 1 Up-regulated stress-related genes DB718607 Chaperone protein ClpB1-like AK319894 Heat shock protein 83-like isoform 1 GO374968 Late embryogenesis abundant protein AK322252 DnaJ protein homolog AK328302 Catalase isozyme 3-like AK247408 Glutaredoxin-family protein AI896522 Glutathione S-transferase L3-like isoform 1 AK330114 Thioredoxin-like 1–2, chloroplastic-like AK329281 4-coumarate–CoA ligase-like 10 AK319251 S-adenosylmethionine decarboxylase proenzyme-like AK324617 Nitrate transporter 1.4-like AK320589 Sodium/calcium exchanger family protein (NCX) AK328035 Probable anion transporter 6, chloroplastic-like TA41929_4081 Aquaporin Membrane intrinsic protein 1;2 AK324676 Sodium transporter HKT1-like DB706854 Aquaporin TIP2-1-like BF096870 Probable sodium-coupled neutral amino acid transporter Up-regulated genes related to hormones biosynthesis/signalling DQ335097 Phytoene synthase 1 AK320933 Abscisic acid (aba)-deficient 4 protein AK328092 1-aminocyclopropane-1-carboxylate oxidase homolog TA41831_4081 Abscisic acid receptor PYL9-like Down-regulated genes related to hormones biosynthesis/signalling AW648744 IAA-amino acid hydrolase ILR1-like 4-like AJ271093 Allene oxide synthase AW041529 Auxin-binding protein ABP19a-like U37840 Lipoxygenase AJ785329 Gibberellin-regulated protein 4

progressively being identified, such as the Mediator Med25 cofactor protein (Blomberg et al. 2012). Among other features of DREB2 regulators, it is also worth noting that most of the ERF encoding genes are intronless, while some members of the group VII display intron in the 5′ regulatory region (Nakashima et al. 2000; Nakano et al. 2006). A large intronic sequence has been identified in SlDREB2 5′ UTR region. It regulates spatial gene expression during germination and early tomato seedling development, but also in response to salinity (Fig. 1c,d). In addition, it enhances SlDREB2 expression, as visible by GUS-staining intensity. 5’UTR introns are classically important in size and are © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

32.8 7 6.9 5 4 3.4 3.3 2.5

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Table 1. Non-exhaustive list of genes differently regulated (P ≤ 0.05) between SlDREB2-transgenic line D4 and WT tomato plants

11.2 9 7.6 7 6.3 4.1 3.1 3 2.8 16 10.3 9.3 6.6 11.4 5.9 5.6 3 9 3.2 5.8 4.9 4.8 4.7 4.6 4.3 4.2 5.9 5.4 3.1 3 23.5 9.3 5.3 5.2 5.1

located close to the initiating ATG codon, and can influence localization and level of gene expression, termed intronmediated enhancement (Chung et al. 2006). 5’UTR introns can besides interfere with stress response (Khurana et al. 2013).

SlDREB2 is involved in ABA and additional hormonal signalling/biosynthesis pathways SlDREB2 is rapidly induced by ABA (Fig. 4b) and 35S::SlDREB2 transgenic seeds presented considerable germination difficulties. Transgenic tomatoes seedlings tend to

76 I. Hichri et al. accumulate more ABA and SA than WT plants. The ABA 8′-hydroxylase encoding gene is likewise down-regulated in D1, D4 and D5 (Fig. 4d), indicating that ABA catabolism is reduced in transgenic lines. Germination impairment of transgenic seeds may be illustrated by this hormonal imbalance, as ABA inhibits germination and seedling establishment (reviewed in Finkelstein et al. 2002), and SA inhibits seed germination as well (Rajjou et al. 2006). In Arabidopsis, DREB2C overexpressing lines are only ABA hypersensitive during germination and early stages of seedling development. DREB2C, similarly to DREB2A, interacts with the basic leucine zipper TFs ABF2, ABF3 and ABF4, known to regulate ABA transduction pathway (Lee et al. 2010). Phylogenetic relationship between AtDRE2BC and SlDREB2 suggests that SlDREB2 may control in tomato comparable mechanisms of ABA biosynthesis/signalling regulation during germination and seedling establishment steps. Plant adaptive response to harsh environmental conditions is tightly controlled by ABA (reviewed in Yoshida et al. 2014). Under control conditions, 35S::SlDREB2 plants grown in hydroponics showed significantly higher ABA contents than in WT and after salinity treatment, ABA contents increased only in transgenic plants (Fig. 6d). Several ABA metabolism-related genes were induced in transgenic plants relatively to WT (Table 1, Fig. 7b). Phytoene synthase catalyses indeed the first committed step in carotenoid biosynthesis, from which ABA is derived. ABA receptor PYL9-like encoding gene was up-regulated as well, suggesting de novo ABA biosynthesis, and subsequent signalling. Finally, the ABA-deficient 4 (ABA4), encoding a neoxanthin synthase, is also up-regulated and may participate to ABA metabolism and plays a role in oxidative stress response (Dall’Osto et al. 2007). Together, our transcriptomic and biochemical data strongly support that SlDREB2 is involved, either directly or not, in ABA biosynthesis and signalling in tomato. A similar pattern of accumulation was described for JA. JA contents increased up to ∼4 times in transgenic plants following salt stress (Fig. 6d). JA-related genes such as JAZ1 repressor but also the lipoxygenase were down-regulated in transgenic plants as early as 2 d after stress onset (Table 1), suggesting a possible negative regulation feed-back loop. In the same manner, tomato MYC2 TF was inhibited in D4 and in Arabidopsis, MYC2 is known to coordinate JA signalling pathway by fine-tuning both positive and negative signalling cascades (Kazan & Manners 2013). In barley, JA pretreatment prior to salinity imposition improved stress response (Walia et al. 2007). Halophytes such as Cakile maritima and Thellungiella salsuginea analogously accumulated more ABA and JA than A. thaliana within short-term salt stress (Ellouzi et al. 2014), emphasizing the importance of JA in stress management. In contrast to ABA and JA, SA contents increased only in WT upon salt stress. Some discrepancies regarding SA mode of action during salinity response have been reported. Indeed, SA treatment improved salt tolerance in wheat seedlings by enhancing expression of antioxidative stress machinery genes (Li et al. 2013), and importance of SA

in salt-stress alleviation has been recently reviewed (Miura & Tada 2014). On the other hand, in germinating Arabidopsis seedlings, SA stimulated oxidative stress generated by salinity (Borsani et al. 2001). These data indicate that SA effects strongly depend on its concentration as well as on plant developmental stage.

SlDREB2 mediates salinity response Salt treatment strongly and constantly increased SlDREB2 expression in young leaves and seedlings cotyledons (Figs 1 and 3), reinforcing the hypothesis of SlDREB2 involvement in protective mechanisms establishment in the photosynthetic organs. Comparable results were reported for AtDREB2A, B and C, where its promoter activity strongly increased in Arabidopsis leaves following NaCl treatment (Nakashima et al. 2000; Lee et al. 2010). Salt tolerance is based on different mechanisms such as ion exclusion or compartmentalization, compatible solutes accumulation and reactive oxygen species (ROS) scavenging, in parallel with physiological and morphological adaptation visible by plant growth inhibition and root/shoot ratio increase, photosynthesis and transpiration retention (Lovelli et al. 2012). Longterm salinity treatment (2 weeks, 125 mm NaCl) significantly increased proline concentration in 35S::SlDREB2 Arabidopsis (Fig. 3). In tomato, reverse tendencies were observed, and transgenic plants showed before stress initiation significantly less proline than in WT. Salinity increased proline accumulation in all types of plants, but this level remained lower in 35S::SlDREB2 plants than in WT 5 d after stress onset, suggesting that different biochemical responses to salt stress occur in Arabidopsis and tomato. Transgenic tomatoes accumulated less osmoprotectants under salinity than WT plants, as also demonstrated by their osmotic potential (Fig. 5b). The wild tomato salt tolerant S. pimpinellifolium accumulates as well less osmoprotectants such as proline and soluble sugars than the cultivated tomato ‘Moneymaker’ (Sun et al. 2010), demonstrating that proline accumulation is actively required in more sensitive varieties. Similarly, PAs are essential signalling molecules involved in oxidative stress alleviation, retention of plant photosynthesis efficiency, ion homeostasis regulation, and modulation of expression of different genes related to osmotic stresses response (Kasukabe et al. 2004; Hazarika & Rajam 2011). Like SlDREB2 transgenic tomatoes, the salt-tolerant S. lycopersicum cv. Jinpengchaoguan accumulates less spermine than a salt-sensitive cultivar (Hu et al. 2012). A lower accumulation of protecting proline and spermine in 35S::SlDREB2 may be related to an improvement of K+ versus toxic Na+ accumulation in these plants comparatively to WT. Several types of transporters that may contribute to osmoregulation by reduction of ion uptake, ion extrusion and compartmentalization were differentially expressed between WT and transgenic plants (Table 1). In particular, the sodium/calcium exchanger (NCX), the high-affinity potassium and sodium transporter HKT1-like, and the sodiumcoupled neutral amino acid transporters (Fig. 7b) may © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 62–79

SlDREB2 mediates salt tolerance in tomato contribute to alleviate the toxicity caused by excessive Na+. Indeed, HKT1 controls Na+ and K+ uptake in Arabidopsis roots (Rus et al. 2001) and Arabidopsis NCX-like atncl mutants display reduced sensitivity to salt in comparison with WT (Wang et al. 2012). Multiple aquaporins encoding genes were also up-regulated following salt stress in 35S::SlDREB2 plants (Table 1) and may as well take part in ionic homeostasis as observed in soybean (Zhou et al. 2014) or the halophyte plant Thellungiella salsuginea (Wang et al. 2014), together with other non-typical transporters, such as the nitrate transporter 1.4-like (Karim et al. 2005). Nitrate transporter 1.4-like belongs to the peptide transporters (PTR), also known as proton-dependent oligopeptide (POT) family, and is involved in nitrogen metabolism. In Arabidopsis, AtPTR3 is strongly induced by salt, and ptr3 KO mutants showed reduced growth and germination frequency on saltcontaining medium, in comparison with WT, reflecting a possible role of PTR3 in salt stress alleviation (Karim et al. 2005). In summary, SlDREB2 was first identified as a salt stressregulated transcription factor, and its overexpression in Arabidopsis and tomato imparted plant tolerance to salinity. This tolerance was due to the efficient regulation of protecting molecules (proline, polyamines) synthesis and hormonal contents (especially ABA), and the regulation of down-stream genes involved in stress response, osmoregulation and hormones biosynthesis/signalling, among others (Supporting Information Fig. S7).We also showed in this study for the first time the important role of an intronic sequence located in the SlDREB2 5’UTR in spatio-temporal gene expression and stress response. In the future, it will be interesting to determine if SlDREB2 can improve plant tolerance to heat or drought as well, as suggested by our experiment (Fig. 5a). In parallel, we specifically established in this study that SlDREB2 was also necessary to different aspects of plant development. In particular, SlDREB2 regulates plant general growth, as early as from germination and post-germination steps, probably in an ABA-dependent way.Transgenic flowers failed to blossom normally because of sepal fusion and fruit formation was affected as well. Further investigations will be needed to clarify SlDREB2 function in flower and fruit establishment, and in-depth analysis of key target genes expression.

ACKNOWLEDGMENTS This work was supported by the Fonds National pour la Recherche Scientifique (FNRS, Belgium, convention N° 1.5117.11), the Czech Science Foundation (P506/11/0774, to E.Z., P.I.D. and V.M.) and the Wallonie-Bruxelles International program (Rhéa 2011/35047). The authors are also thankful to B. Vanpée, J. Bar and T. Dagbert for technical assistance, as well as to Abdelmounaim Errachid for his help with confocal microscopy.

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Received 3 March 2015; received in revised form 17 May 2015; accepted for publication 18 May 2015

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Amino acids sequence comparison of SlDREB2 and its closest orthologs SlDREB1, AdERF3, SbDREB2A and AtDREB2C. Figure S2. T-DNA insertion in Arabidopsis (A) or tomato (B) genomes and expression of SlDREB2 in transgenic and WT lines were analysed by PCR.

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Figure S3. Germination percentage of 35S::SlDREB2 (L4, L5 and L11) and WT lines on control (half-strength MS) medium. Figure S4. Phenotype (5 d after sowing) of WT, atdreb2a, V and SlDREB2-complemented lines on control medium or media supplemented with NaCl. Figure S5. Photosynthesis parameters of SlDREB2 transgenic and WT tomato plants exposed to control conditions or salt (125 mm NaCl) for 10 d. Figure S6. SlDREB2 overexpression enhances tomato salt tolerance (Independent Experiment 2). Figure S7. Summary of SlDREB2 mode of action during tomato seeds germination and plant growth, and in response to osmotic stresses. Table S1. Gibberellins contents in Leaf 4 of transgenic (D1, D4 and D5) and WT plants [control (0) or exposed for 5 d to salinity (5)]. Table S2. Sequence of primers used for cloning and qRTPCR analyses.