Plant Cell Rep DOI 10.1007/s00299-015-1825-6

ORIGINAL ARTICLE

The promoter of the AlSAP gene from the halophyte grass Aeluropus littoralis directs a stress-inducible expression pattern in transgenic rice plants Rania Ben-Saad3 • Donaldo Meynard2 • Walid Ben-Romdhane1,3 • Delphine Mieulet2 Jean-Luc Verdeil2 • Abdullah Al-Doss1 • Emmanuel Guiderdoni2 • Afif Hassairi1,3

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Received: 20 March 2015 / Revised: 27 May 2015 / Accepted: 12 June 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Key message When fused to ‘‘PrAlSAP’’ promoter, transcripts of gusA exhibited similar accumulation patterns in transgenic rice as AlSAP transcripts in A. littoralis. PrAlSAP can be used for engineering abiotic stress tolerance. Abstract We previously showed that ectopic expression of a stress-associated protein gene from Aeluropus littoralis (AlSAP) enhances tolerance to multiple abiotic stresses in tobacco, wheat and rice. The ortholog of AlSAP in rice is OsSAP9. Here, we demonstrate that AlSAP transcripts accumulate in Aeleuropus in response to multiple abiotic stresses and at a higher level in roots, while those of OsSAP9 are preferentially induced by cold and heat treatments and accumulate preferentially in leaves of rice. In silico analysis of the AlSAP promoter ‘‘PrAlSAP’’ predicted several cis-acting elements responsible for gene regulation by dehydration, salt, heat, ABA, SA, wounding and tissueCommunicated by L. Pen˜a. R. Ben-Saad and A. Hassairi should be considered as first authors, as they contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00299-015-1825-6) contains supplementary material, which is available to authorized users. & Afif Hassairi [email protected] 1

Present Address: College of Food and Agriculture Sciences, King Saud University, Po Box 2460, Riyadh 11451, Saudi Arabia

2

CIRAD, UMR AGAP, 34398 Montpellier Cedex 5, France

3

Centre of Biotechnology of Sfax (CBS), University of Sfax, LPAP, Po Box 1117, 3018 Sfax, Tunisia

specific expression. The PrAlSAP promoter was fused to the gusA gene and used to produce transgenic rice plants. Transcripts of gusA exhibited similar accumulation patterns in transgenic rice as AlSAP transcripts in A. littoralis. Indeed, accumulation of gusA transcripts was higher in roots than in leaves and induced by salt, drought, cold and heat treatments. GUS activity was confirmed in roots, coleoptiles, leaves and glumes, but absent in the root cell elongation zone and in dry seeds. A wound treatment strongly induced GUS accumulation in leaves and imbibed seeds. Altogether, these results indicate that the regulatory regions of two ortholog genes ‘‘AlSAP’’ and ‘‘OsSAP9’’ have diverged in the specificity of the signals promoting their induction, but that the trans-acting elements allowing the correct spatiotemporal regulation and stress induction of PrAlSAP exist in rice. Therefore, the AlSAP promoter appears to be an interesting candidate for engineering abiotic stress tolerance in cereals. Keywords Abiotic stress  Aeluropus littoralis  AlSAP gene  OsSAP9 gene  AlSAP promoter ‘‘PrAlSAP’’transgenic rice

Introduction Abiotic stresses impose serious threat to productivity and sustainability of cereals worldwide. To improve stress tolerance in crop plants, a large range of genes, including transcription factors, has been engineered under the control of constitutive or stress-inducible promoters. While the promoter regions of the CaMV35S (Odell et al. 1985), rice Act1 (McElroy et al. 1990) and maize Ubi1 (Christensen et al. 1992; Cornejo et al. 1993) genes have been extensively used to drive high and constitutive expression of

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transgenes conferring abiotic stress tolerance, experience has also highlighted that the continuous and ubiquitous expression of some of these transgenes may be detrimental to plant growth and yield. Phenotypic alterations such as low fertility, growth retardation, abnormal development and reduced seed production have been reported in overexpressers (Capell et al. 1998; Hsieh et al. 2002; Karim et al. 2007; Kasuga et al. 1999; Nakashima et al. 2007; Romero et al. 1997). In addition, accumulation of proteins encoded by the transgenes at a time and level not needed for their action can lead to excessive energy consumption causing phenotypes that are not directly correlated with the recombinant protein itself (Himmelbach et al. 2007; Hsieh et al. 2002; Kasuga et al. 1999). To avoid such unwanted pleiotropic effects in engineering abiotic stress tolerance, well-characterized tissue-specific and abiotic stress-inducible promoters are needed in plant biotechnology. Several stress-inducible promoters have already been characterized including OsABA2 (Rai et al. 2009) and Arabidopsis rd29A (Yamaguchi-Shinozaki et al. 1990). The biotic and abiotic stress-inducible rd29A promoter was widely used in plant transformation to minimize the negative effects on plant growth in A. thaliana, tobacco, wheat, sugarcane, potato and rice plants (Behnam et al. 2006; Bihani et al. 2011; Gao and Ma 2005; Kasuga et al. 1999; Sun 2002; Wu et al. 2008; Zhang et al. 2004). Nevertheless, most stress-inducible promoters available to date are derived from dicotyledonous plants with the exception of few rice and barley drought-inducible promoters (Xiao and Xue 2001). Isolation and characterization of other stressinducible promoters allowing a conditional expression of transgenes, functionally verified in cereals and other monocotyledonous species, is therefore needed. It has been proposed that, besides displaying remarkable morphological and anatomical adaptations, halophytes largely use similar mechanisms of salt tolerance than glycophytes. It has been also hypothesized that the contrasted tolerance observed between glycophytes and halophytes may result from differences in the regulation of the underlying response pathways (Taji et al. 2004). Therefore, beyond the use of halophytes as a source of coding sequences and proteins for engineering stress tolerance in glycophyte plants, it is worth exploring them as a valuable source of regulatory sequences. Among the families of stress response genes attracting interest, the ‘‘STRESS ASSOCIATED PROTEIN’’ (SAP) family includes genes encoding proteins having A20 and AN1 zinc finger domains that are induced by one or more abiotic stresses and perform their functions in a stressspecific and/or tissue-specific manner (Ben-Saad et al. 2010, 2012a, b, c; Jin et al. 2007; Vij and Tyagi 2006, 2008).

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Overexpression of the SAP genes from rice and Arabidopsis has consistently conferred enhanced tolerance to abiotic stresses in homologous or heterologous systems (Giri et al. 2011; Huang et al. 2008; Kang et al. 2011; Kanneganti and Gupta 2008; Mukhopadhyay et al. 2004). We have recently isolated and characterized the AlSAP gene belonging to the A20/AN1 SAP family from the C4 halophyte grass A. littoralis. This gene has shown to be induced by salt, drought, cold, heat, ABA and SA. Furthermore, we have demonstrated that the expression of AlSAP under the control of a constitutive promoter in tobacco, durum wheat and rice plants improved their tolerance to continuous salt and drought stresses under greenhouse conditions (Ben Saad et al. 2010, 2012a, b, c). Rice contains 11 genes belonging to the A20/AN1 SAP family and OsSAP9 appears to be the closest ortholog of AlSAP, sharing 75 % amino acid identity. However, it was found that the expression of OsSAP9 increased the tolerance to both high and low temperature and H2O2 stresses, but conferred an oversensitivity to dehydration and salt stresses in tobacco plants (Huang et al. 2008). This would suggest that AlSAP and OsSAP9 accomplish different functions in their respective hosts, OsSAP9 having evolved in a more specialized role in thermal stress tolerance. However, overexpression of OsSAP9 was realized in a heterologous host, tobacco, and may have also led to a deregulation of other endogenous SAP genes. We compared the regulation of the putative orthologous genes AlSAP and OsSAP9 in their respective hosts, the C4 halophyte grass A. littoralis and the C3 glycophyte cereal rice, respectively. Furthermore, to determine whether rice contains the trans-acting elements allowing a correct regulation of the AlSAP promoter, we isolated and cloned a sequence upstream of the AlSAP translated region, hereafter referred to as the PrAlSAP promoter, fused it to the gusA coding sequence and introduced it into rice. The same promoter sequence has been recently shown to be active in tobacco plants in an age-dependent, multiple abiotic stressinducible and tissue-specific manner (Ben Saad et al. 2011). We investigate hereafter the spatiotemporal activity of the PrAlSAP promoter in stably transformed rice plants using both q-PCR and immuno- and histochemical assays. Our results show that PrAlSAP is active in rice and exhibits the same spatiotemporal regulation and induction by multiple abiotic stresses than that observed with AlSAP transcripts in Aeluropus. Also, it was demonstrated that the PrAlSAP promoter was induced by wounding. This work indicates that the PrAlSAP is the first promoter isolated from the halophyte C4 grass Aeluropus littoralis and could be useful for driving stress-responsive, spatiotemporal accumulation of proteins conferring tolerance to abiotic/biotic stresses in transgenic cereal crops.

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Materials and methods Plant materials Seeds of Aeluropus littoralis (autogamy plant) were collected from salt marshes near ‘‘Sfax’’, a coastal town in the middle of Tunisia. For rice transformation, seeds of Oryza sativa L. japonica cv. ‘Nipponbare’ were used and originally provided by the National Institute of Agro-biological Sciences (NIAS, Tsukuba, Japan). In silico analysis of PrAlSAP and PrOsSAP9 To identify the promoter of the OsSAP9 gene, the ‘‘Rice Annotation Project Database’’ (RAP-DB) was used. The genomic structure of the OsSAP9 gene was identified first, and then 1000-bp upstream its 50 UTR were selected and designed as the PrOsSAP9 promoter. To compare the cisacting elements existing in the two promoters (PrAlSAP and PrOsSAP9), their respective sequences were analyzed using two databases search programs, PlantCARE database (Lescot et al. 2002; Rombauts et al. 1999) and PLACE database (Higo et al. 1999). Construction of binary vector and rice transformation A 586-bp genomic DNA fragment upstream of the 50 region of the AlSAP translated sequence, including a 83-bp 50 UTR sequence (AlSAP promoter: PrAlSAP), a putative transcription start site (TSS: ?1) at position 371 and a TATA-box at position -30 (relative to TSS), was released by SpeI/NcoI from pGEM–PrAlSAP then cloned upstream the gusA gene into the pCAMBIA1301 vector (CAMBIA, Canberra, Australia) and digested with XbaI/NcoI to substitute the CaMV-35S promoter. Following the same method, the AlSAP promoter lacking the 83-bp 50 UTR sequence (AlSAP promoter minus the 50 UTR: PrAlSAP–50 UTR) was released from pGEM–PrAlSAP–50 UTR then cloned upstream the gusA gene into the pCAMBIA1301 vector. The resulting constructs, pCAMBIA1301–PrAlSAP–gusA, pCAMBIA1301– PrAlSAP–50 UTR–gusA and the pCAMBIA1301–CaMV35S– gusA were then mobilized into Agrobacterium tumefaciens EHA105 strain (Hood et al. 1993) by the freeze–thaw transformation method (Chen et al. 1994). The two constructs were used to transform embryogenic calli prepared from rice mature seed embryos (Sallaud et al. 2003). The regenerated hygromycin-resistant plants, named PrAlSAP– gusA, PrAlSAP–50 UTR–gusA and 35S–gusA, were cultivated on MS agar medium containing 50 mg/l hygromycin. The transgenic T0 plants were transplanted into soil and allowed to self-fertilize for producing seeds of T1 and then T2

generation. T2 homozygous plants were used in the subsequent assays. The WT rice and 35S–gusA transgenic plants were used as negative and positive controls, respectively. Southern and northern blot analyses The integration and expression of the gene cassettes were ascertained by Southern and northern blot hybridization, in T0 and T2 generations, respectively. For Southern blot, genomic DNA was extracted from leaves using mixed alkyl trimethyl ammonium bromide (MATAB) buffer [100 mM Tris–HCl, pH 8.0, 1.5 M NaCl, 20 mM EDTA, 2 % (w/v) MATAB, 1 % (w/v) PEG 6000, 0.5 % (w/v) sodium sulfite Na2SO2]. A total of 1 g of leaves was ground in liquid nitrogen and immediately transferred to 9 ml of 74 °C pre-warmed extraction buffer. Crude extracts were maintained for 20 min at 74 °C and then cooled to room temperature. Equal volumes of chloroform– isoamylalcohol (CIAA) 24:1 were added and mixed by inversion. The mixture was then centrifuged at 5000 rpm for 30 min. The supernatant was transferred into a new clean Falcon tube, treated with RNase A for 30 min and extracted once more with 9 ml CIAA as shown above. Supernatant containing DNA was collected in 50 ml Falcon tube containing 9 ml of absolute isopropanol and centrifuged at 7000 rpm. Ethanol 70 % was used to wash the pellet and the DNA samples were then hydrated with 1000 ll of TE buffer. The concentration of the DNA was measured with spectrophotometer at an absorbance of 260/280 nm. The quality of DNA was checked on 0.8 % agarose gel. Twenty micrograms of genomic DNA was digested overnight with NcoI. The digested DNA was separated by electrophoresis on a 1 % agarose gel and then transferred onto Hybond-N? nylon membrane (AMERSHAM-BIOSCIENCES). The gusA cDNA fragment amplified by PCR with a pair-specific primer (GUSF and GUSR) and labeled with [a-32P]dCTP (AMERSHAMBIOSCIENCES) was used as a probe. After hybridization at 65 °C, the membrane was washed once with 2X SSC plus 0.1 % SDS at 65 °C for 20 min and then twice with 1X SSC plus 0.1 % SDS at 65 °C for 30 min. For RNA blot analysis, total RNA was extracted from 1-month-old seedlings of transgenic rice using the RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s recommendations. The residual amount of DNA remaining in RNA was removed using on-column DNase I (PROMEGA) treatment during the RNeasy procedure. About 20 lg of total RNA samples was resolved on a 1.5 % formaldehyde gel and blotted onto a Hybond-N? membrane. Hybridization and washing conditions were identical to those of the above-mentioned DNA blot. Hybridization was detected by autoradiography.

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Abiotic stress treatments The expression profiles of the two orthologous genes, AlSAP and OsSAP9, were established in stressed A. littoralis and rice plants, respectively. Surface-sterilized seeds of A. littoralis (1 % sodium hypochlorite solution for 15 min, followed by six washings with sterile distilled water) were germinated in Eppendorf tubes containing 500 ll half-strength MS (Murashige and Skoog 1962) solid medium under 16/8 h photoperiod at 25 °C. The tubes containing the seedlings were perforated and their caps cut when the plant roots reached their bottom. Later, the seedlings were transferred to a nutrient solution as described by (Zouari et al. 2007) and grown in greenhouse condition before stress treatments. For rice, healthy seeds of WT (wild-type) Nipponbare rice were treated with 70 % ethanol for 1 min, rinsed with sterile distilled water, treated with 1 % sodium hypochlorite solution for 20 min and rinsed three times again with sterile distilled water. The sterilized seeds were germinated in Petri dishes containing imbibed Whatman filter paper with 10 ml sterile distilled water. The dishes were then incubated in a growth chamber maintained at day/night temperature of 28/25 °C and a 16/8 h photoperiod. After 1 week, the germinated seedlings were transferred to 100 ml beakers and grown for another 1 week before stress application. The stress factors used are: salt stress (150 mM NaCl), osmotic stress (10 % PEG 8000), low temperature (4 °C) and high temperature (42 °C). Following each treatment, leaves and roots from A. littoralis and rice plants were sampled at 12, 24, 48 and 72 h, frozen in liquid nitrogen and stored at -80 °C for RNA extraction and qPCR reactions. Seeds were collected for histochemical GUS staining. The whole transgenic grains or seedlings were stained before germination and 12, 48 and 72 h following germination. Plants were stressed for 48 h with different abiotic stresses 6 DAG (days after germination): 150 mM NaCl, 125 mM mannitol, 42 °C heat, 100 lM ABA, 10 mM SA, 100 lM MejA and 4 °C. To determine whether AlSAP promoter activity is altered in response to wounding, old leaves of mature transgenic rice plants and seeds were mechanically wounded with a blade and stained 1 h after wounding with GUS assays. Histochemical GUS staining Histochemical staining of GUS activity was performed by vacuum infiltrating tissue sections or seedlings in 50 mM Na2HPO4 buffer (pH 7.0), 0.5 mM K3(Fe[CN]6), 0.5 mM K4(Fe[CN]6), 0.1 % Triton X-100 and 1 mg/l X-Gluc (5-bromo-4-chloro-3-indolyl b-D-glucuronide cyclohexylammonium salt) and incubating them at 37 °C overnight (Jefferson et al. 1987). Before photographing or

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histological analyzing, the stained tissues were soaked in 70 % ethanol to remove the pigments and chlorophyll. Tissue observations were performed with a Leica MZFLIII binocular microscope (Leica Microsystems, Heerbrugg, Switzerland). For histological sectioning by microtome, the tissues were fixed during 30 min at room temperature in [200 mM Na2HPO4 buffer (pH 7.0) containing 10 % (v/v) paraformaldehyde, 2 % (v/v) glutaraldehyde, and 50 mM caffeine]. To prepare transversal sections of 3 lm, a microtome was used after dehydrating and embedding the same organs in molds with resin. Dehydration was performed in a graded ethanol series (70–95 %, v/v), followed by a final butanol bath. Then, they were impregnated in a Technovit 7100 resin (Kulzer, Wehrheim, Germany)-butanol (1:1, v/v) mixture. Finally, tissues were embedded in molds with resin and polymerized for 2 h by adding the polymerization agent to the resin. The observations of tissue sections prepared by microtome were carried out with a Leica DMRXA fluorescence microscope (Leica Microsystems) under dark-field light. Quantification of mRNA expression by qPCR analysis Transcript levels of the AlSAP, OsSAP9 and gusA genes were monitored in 60-day-old Aeluropus seedlings, 7-dayold rice cv Nipponbare seedlings and 7-day-old T2 homozygous PrAlSAP:gusA seedlings treated with NaCl, PEG, cold and heat for 12, 24, 48 and 72 h. The time difference at the initiation of the experiment is due to the slower growth of Aeluropus seedlings compared to rice seedlings under control conditions. Total RNA was isolated with the RNeasy Plant Mini Kit (QIAGEN) from 200 mg of leaf or root materials according to the manufacturer’s recommendations. The residual amount of DNA remaining in RNA was removed using on-column DNase I (PROMEGA) treatment during the RNeasy procedure. Firststrand cDNA synthesis was performed on 2 lg RNA using SuperScriptTM II Reverse Transcriptase (INVITROGEN) with oligo (dT) 18 and random hexamer primers according to the manufacturer’s instructions. Primer pairs were designed to amplify fragments of AlSAP, OsSAP9, gusA and housekeeping OsExp, rRNA26s genes with Primer 3 software to ensure gene specificity (Rozen and Skaletsky 1999). The following primers were used for real-time qPCR: AlSAPF, AlSAPR, 26SF and 26SR (for Aeluropus littoralis); OsSAP9F, OsSAP9R, OsExpF and OsExpR (for Oryza sativa); and GUSAF and GUSAR (for transgenic rice) (supplementary Table 1). The reverse transcriptase (RT) reactions were diluted 1/5th and used as a template in real-time qPCR reactions. The amplification reactions were performed in 15 ll final volumes containing 7.5 ll of 2 9 Quantitect SYBER

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Green I mixture (QIAGEN, Germantown, USA), 1.5 ll of primer-pair mix (0.5/0.5 lM for forward and reverse primers), 3 ll of cDNA and 3 ll of RNase-free water. Reactions were carried out in Light-Cycler 480 (ROCHE, Basel, Switzerland). Thermal cycling conditions were 5 min at 95 °C, followed by 45 cycles of 20 s at 95 °C, 15 s at 60 °C and 20 s at 72 °C. Each sample reaction was set up in triplicate to ensure the reproducibility of the results. cDNAs to be amplified (target and reference) were made with the same PCR master mix. Melting curve analysis at the end of cycling was used to verify that there was single amplification. At the end of the reaction, the threshold cycle (CT) values of the triplicate PCRs were averaged and used for transcripts’ quantification. The relative expression was quantified by using the comparative CT method with the rRNA26s or OsExp gene as an internal expression standard (Livark and Schmittgen 2001). The relative expression level was calculated as follows: 2-DDCT , where DDCT = (CT, Target gene - CT, osExp or rRNA26S) stressed - (CT, Target gene - CT, osExp or rRNA26S)control. The relative expression ratios from three independent experiments (three biological repetitions) are reported.

source without sucrose) under 50 rpm gentle shaking and 12:12 h light/dark regime for 7 days. The developing seedlings were then transferred into flasks containing either MS medium or MS medium supplemented with 10 % PEG 8000 for 48 h further growth before tissue collection. The fluorometric assay for specific GUS enzyme activity was quantified in leaf tissues and conducted by measuring the hydrolysis rate of the fluorogenic substrate 4-methylumbelliferyl b-D-glucuronide (MUG) as described in Jefferson et al. (1987). The concentration of protein extracted in the GUS assay buffer (50 mM sodium phosphate, pH 7.0; 10 mM b-mercaptoethanol; 10 mMEDTA, and 0.1 % Triton X-100) was determined according to Bradford (1976) using a Multiskan (Labsystems) spectrophotometer. The GUS specific activity (hydrolysis of 4-methylumbelliferone glucuronide per mg protein per min) was determined using a Fluoroscan (Labsystems) apparatus operating at 460 nm emission wave length.

Results

Immunoblotting detection of GUSA

Various abiotic stress treatments induce AlSAP and OsSAP9

Total proteins were extracted from leaf and root tissues of transgenic rice (PrAlSAP–gusA, PrAlSAP–50 UTR–gusA or CaMV35S–gusA), together with their WT controls, in the extraction buffer [50 mM Tris–HCl (pH 8), 10 mM MgCl2, 1 mM EDTA 0.5 M (pH 8), 5 % glycerol, 1 mM DTT, 0.1 % Triton X-100 and 1 mM phenylmethylsulphonylfluoride (PMSF)] using a rounded glass pestle, followed by centrifugation at 13,000 rpm for 15 min at 4 °C. The protein concentration of the supernatant was determined by the Bradford method (1976) using the Bio-Rad protein assay as described by the manufacturer. Fifty micrograms of total soluble proteins were blotted (iBlotTM Gel Transfer Stack) onto a nitrocellulose membrane (INVITROGEN). GUSA protein was detected following the manufacturer’s instructions of the SNAPi.d method (SNAPi.d., protein detection systems, blots holders, MILLIPORE) and using as primary antibody (at 1:10.000 dilution) the anti-GUS rabbit IgG (H ? L) fraction (Molecular Probes, Eugene, OR) and as the secondary antibody (at 1:20.000 dilution) anti-rabbit IgG horseradish peroxidase conjugate (SIGMA). The blots were washed three times in 1x TBS buffer with 0.1 % Tween-20 and 1 % BSA and developed using the ECL plus Western blotting detection system (AMERSHAM). Surface-sterilized seeds of Nipponbare and T2 transgenic homozygous lines (PrALSAP:gusA, line NB2.3; CaMV35S:gusA, line 9.3) were germinated in flasks containing liquid MS medium (MS salts, vitamins and iron

The promoter regions of PrAlSAP and PrOsSAP9 were analyzed for the presence of putative regulatory motifs using the PLACE and PlantCARE databases. In both promoters, several basic putative cis-acting regulatory elements were predicted, such as the TATA-box, CAAT-box and 50 UTR Py-rich stretch (see supplementary Table 2). In addition, the two promoters present a common large number of ciselements related to meristem-, root-, guard cell-, vasculartissue- and mesophyll-specific expression. Moreover, in both promoters, other common motifs related to abiotic, biotic and hormonal stresses were revealed, such as those involved in the response to dehydration, salt, heat, low temperature, ABA, pathogen and fungal elicitor. However, some cis-acting elements are specific to PrAlSAP such as the ones induced by salicylic acid (SA) and wounding, while some other cis-acting regulatory elements involved in the MeJA and auxin responsiveness are specific to PrOsSAP9. The full list of cis-acting elements found in the two promoters may be viewed in supplementary Table 2. Real-time qPCR analysis was used to evaluate the response to abiotic stresses of the AlSAP and OsSAP9 genes, encoding putatively orthologous A20/AN1 zinc finger proteins, in Aeluropus littoralis and rice, respectively. RNA was isolated from leaves and roots of Aeluropus littoralis and rice plants following stress application during 12, 24, 48 and 72 h, either by salt (150 mM NaCl), osmotic (10 % PEG8000), cold (4 °C) or heat (42 °C) treatments. To gain insight into the basal

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expression levels of the AlSAP and OsSAP9 genes, the threshold cycle values (CT) of the unstressed samples (after normalization with rRNA26S and OsExp gene, respectively) were compared to each other. The transcripts of AlSAP and OsSAP9 genes (relative to the unstressed control) showed an increase in response to all the applied stress treatments. The expression level of AlSAP was higher in roots than in leaves under all abiotic stress treatments (Fig. 1). When treated with 150 mM NaCl, AlSAP transcript accumulation peaked at 48 h in both roots and leaves and then declined. In the case of cold stress, the expression level increased gradually as a function of exposure time of A. littoralis leaves and roots to the treatment. Under heat and osmotic stresses, the induction reached a higher level in roots than in leaves in the first 2 days, then decreased on day 3 after treatment. For OsSAP9, a higher induction of transcript level was observed in leaves than in roots under all the abiotic

stress treatments (Fig. 1). When treated with 150 mM NaCl, the expression level of OsSAP9 gene peaked at 48 h in leaves and then declined at 72 h; however, the treatments only slightly induced the gene in roots. Under osmotic treatment, the OsSAP9 gene showed an induction in leaves 12 h following PEG treatment. The most important induction of OsSAP9 was observed upon cold and heat treatments, in both leaves and roots at 24 h. In conclusion, it is clear that AlSAP transcripts accumulation in response to abiotic stresses is more important in roots than in leaves, whereas OsSAP9 has an inverse behavior (Fig. 1). In addition, the maximum relative expression level (REL) under stress conditions in leaves is more than 150-fold for AlSAP, while it is only more than 60-fold for OsSAP9 when compared to control condition (Fig. 1). Finally, the same trend was observed in roots since the maximum REL is more than 300 for AlSAP and only 7 for OsSAP9.

Fig. 1 Comparative expression profile of AlSAP and OsSAP9 gene in response to abiotic stresses in leaves and roots of Aeluropus littoralis and rice. The relative expression level of AlSAP and OsSAP9 genes in roots and leaves was measured by real-time qPCR under control condition, and under salt (150 mM NaCl), osmotic (10 % PEG8000), cold (4 °C) and heat (42 °C) stresses. Total RNA was extracted from

Aeluropus littoralis and rice plants after various treatments. The amounts of cDNAs of Aeluropus littoralis and rice were calibrated using the rRNA26S and OsExp genes, respectively, as internal controls. The results are presented as means and standard errors from three independent experiments

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Fig. 2 Analysis of transgenic rice lines expressing the gusA gene under the AlSAP promoter. a Schematic representation of the T-DNA cloned in the pCambia1301 binary vector and used for rice transformation. PrAlSAP:gusA: the stress-inducible promoter PrAlSAP fused to gusA gene; 35SCaMV–hptII: the selection marker gene hptII fused to 35SCaMV. b Southern blot analysis of transgenic and wildtype rice plants determining the T-DNA copy number. Genomic DNA was digested with NcoI, separated by electrophoresis on a 1 %

agarose gel and transferred onto a nylon membrane. The transferred DNA was hybridized with the gusA gene probe labeled with [a-32P] dCTP. c Northern blot analysis of gusA transcript levels in wild-type and the transgenic lines of rice. RNA was transferred onto a nylon membrane and hybridized with the a gusA gene probe labeled with [a-32P] dCTP. Equal loading in each lane was confirmed by ethidium bromide staining (lower panel)

Generation of PrAlSAP:gusA rice lines

of gusA transcripts was observed in tobacco roots, no GUSA protein and consequently no GUS activity was detected (Ben Saad et al. 2011). To determine whether a similar spatiotemporal pattern was observed in rice, different organs and tissues of T2 PrALSAP:gusA rice plants were subjected to histochemical assays. Though GUS activity was not detected in dry seeds before germination (Fig. 3a), the AlSAP promoter was found to be active from an early stage of seed germination and throughout the seedling stage (Fig. 3a). Intense GUS staining was observed in the germinating embryo axis, coleoptile and seminal root elongation zone 12, 48 and 72 h after germination (Fig. 3a). At day 6 after germination, GUS staining was restricted to the base of the coleoptile and in the vascular tissues of crown roots, revealing a change in AlSAP promoter activity. As expected, no GUS activity was observed in comparable organs of WT used as negative control, while deep blue GUS staining was detected in the same tissues of PrCaMV–35S:gusA seedlings used as positive controls (Fig. 3a, b). Roots of PrAlSAP:gusA seedlings exhibited GUS staining in the root apical meristem and distal part of the primary root as well as at the branching sites of lateral roots (Fig. 3a). The presence of strong GUS activity in roots therefore contrasted with previous observations in PrAlSAP:gusA tobacco roots where gusA transcripts were detected, but no GUS protein or activity. Strong GUS activity was also detected in the leaf lamina joint region. For floral organs, PrAlSAP:gus A expression was observed in the lemma and palea, which are modified leaves, but no expression was detected in stamens and

To analyze the tissue spe ntal regulation of the AlSAP gene, the pCAMBIA1301–PrAlSAP–gusA construct (Fig. 2a) was introduced into O. sativa ‘Nipponbare’ via Agrobacterium tumefaciens co-cultivation. The presence of the PrAlSAP– gusA fusion in transgenic plants was first confirmed by PCR, using gusA primers. The copy number of the integrated gusA gene was determined by Southern blot analysis (Fig. 2b). We selected five transformed rice plants derived from independently transformed cell lines harboring one or two copies of the gusA gene (Fig. 2b). The expression of the gusA gene in the selected transgenic lines was established by northern blot analysis (Fig. 2c). These lines were propagated and grown in greenhouse till seed maturity. The T2 homozygous seedlings and mature plants of a representative transgenic line (line NB2.3) were used to investigate the functional properties of the AlSAP promoter. In that aim, gusA transcript accumulation was monitored by real-time qPCR, and b-glucuronidase activity was detected by histochemical staining in various organs throughout plant development. Spatiotemporal activity of the AlSAP promoter in PrAlSAP:gusA lines We have previously reported that PrAlSAP is an organspecific promoter and drives both developmental and basipetal expression pattern of the gusA gene in the heterologous host tobacco. However, though accumulation

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Plant Cell Rep Fig. 3 Histochemical localization of GUS activity in transgenic rice during seed germination, in flowers and in leaf lamina joint region. a Line NB2.3 PrAlSAP–gusA, CaMV35S–gusA and WT seeds before germination and 12, 48 and 72 h after germination were subjected to GUS histochemical assays. Seeds and seedlings of CaMV35S–gusA and WT lines were used as positive and negative controls, respectively. b GUS activity in flowers and in leaf lamina joint region of lines NB2.3 PrAlSAP–gusA, CaMV35S–gusA and WT rice flowers

pistils. All flower tissues of PrCaMV–35S:gusA plants exhibited deep blue staining, while no GUS staining was observed in wild-type rice flowers (Fig. 3b). The cell specificity of PrAlSAP activity in rice was examined by observation of transversal cross sections of stained tissues in several independent lines. Cross sections of leaf blades from PrAlSAP:gusA seedlings revealed an accumulation of GUS products in the mesophyll, sclerenchyma, bundle sheath and phloem cells, while little or no staining was observed in the upper and lower epidermis, bulliform cells and xylem (Fig. 4a). As anticipated, wild-

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type rice leaf blade tissues did not exhibit any background GUS staining (Fig. 4b). Transversal cross sections of leaf blade of control PrCaMV–35S:gusA plants revealed GUS products in all the leaf and root tissues (Fig. 4c). Sections of root (Fig. 4d) revealed strong GUS activity in the root cap (Fig. 4g), while no GUS product was observed in the zone of cellular division (Fig. 4f). Radial sections in the zone of cellular maturation revealed GUS staining in the epidermis, exodermis, sclerenchyma cortex, endodermis, metaxylem and phloem (Fig. 4e). A similar pattern was observed across different transformation events.

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Fig. 4 Histological localization of GUS activity under dark-field illumination (GUS crystals appear pink) in leaves and roots of NB2.3 PrAlSAP:gusA rice line, CaMV35S:gusA and WT tobacco: Transversal microtome sections through (a) a leaf blade of transgenic rice harboring the PrAlSAP:gusA, b leaf blade of WT rice and c leaf blade

of transgenic rice harboring the CaMV35S:gusA. Transversal microtome sections through the seminal root of transgenic rice harboring the PrAlSAP:gusA, d zone of cellular maturation, e zone of cellular division and f root cap. ue upper epidermis, le lower epidermis, me mesophyll, Ph phloem, Xy xylem, sc sclerenchyma. Bars 30 lm

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Regulation of PrAlSAP in response to abiotic stresses in transgenic rice plants It has previously been reported that expression of the AlSAP gene is induced by abiotic and hormonal stresses in A. littoralis (Ben Saad et al. 2010). Here, histochemical Fig. 5 Induction by abiotic stress treatments of the PrAlSAP promoter in transgenic 2.3 rice line. a Histochemical assays on representative 6 DAG (days after germination) seedlings stressed with NaCl (150 mM), mannitol (125 mM), heat (42 °C), ABA (100 lM), SA (10 mM), MejA (100 lm) and cold (4 °C). b Histochemical assays on representative 6 DAG (days after germination) WT and 35S–gusA seedlings. c Western blot analysis using the anti-GUS antibody was used to detect the presence of GUS protein in leaves and roots exposed to control and stress conditions for 48 h in transgenic rice. WT wild-type rice

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staining showed GUS expression in whole plants harboring the PrAlSAP–gusA construct, 6 days following germination under control, NaCl, mannitol, heat, ABA, SA, MejA and cold conditions (Fig. 5a). Tissues of stress-treated NB2.3 rice seedlings exhibited a much deeper staining than those from seedlings grown under control conditions (Fig. 5a).

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The GUS staining intensity was particularly enhanced in roots upon stress treatment (Fig. 5a). However, when gusA was expressed under control of the constitutive CaMV–35S promoter, a similar dark homogenous GUS staining was observed in the root and leaf (Fig. 5b). Finally, no GUS staining was observed in non-transformed plants. Immunoblot analysis of total soluble protein extracts confirmed the stimulation of GUSA accumulation from a steady-state level in roots and leaves by multiple abiotic stresses (Fig. 5c). In roots, accumulation of gusA by all stresses was stimulated to a higher level than in leaves. No GUSA protein was detected in WT plant (Fig. 5c). It is important to notice that the same results for GUS expression were observed in transgenic seedlings of the line NB10.1 harboring the PrAlSAP–50 UTR–gusA construct (Supplementary Fig. 1). It is clear that the 83 bp of the 50 UTR has no effect on the promoter activity. We further estimated the relative strengths of the PrAlSAP promoter region and of the commonly used CaMV35S promoter in directing GUS accumulation upon induction by abiotic stress treatment. In that aim, we carried a GUS quantitative fluorometric assay in leaf tissues of 7-day-old seedlings further treated or not with PEG 10 % for 48 h. GUS activity in the PrAlSAP:gusA line NB2.3 was monitored along with a CaMV35S:gusA line 9.3 expresser. Steady-state GUS activity under control condition appeared lower in leaves of the PrAlSAP:gusA line NB2.3 than in the line 9.3 CaMV35S:gusA. However, following the 10 % PEG treatment, GUS activity in leaves of the PrAlSAP:gusA line NB2.3 was twofold more than the one in leaves of line 9.3 CaMV35S:gusA (Supplementary Fig. 2). To investigate whether the 586-bp fragment upstream the AlSAP translated sequence is sufficient to maintain its spatiotemporal and inducible regulation in rice, we investigated the accumulation of gusA transcripts in root and leaf tissues of PrAlSAP:gusA plants under various abiotic stress treatments. Seven-day-old T2 seedlings of two independent PrAlSAP:gusA lines (NB2.3 and NB12.1) were treated with salt, PEG, cold and heat and then the total RNA was isolated from their leaves and roots after 12, 24, 48 and 72 h following application of stresses. Real-time qPCR analyses revealed that the gusA transcripts accumulate at a higher level in roots than in leaves under different abiotic stresses. In addition, under NaCl (150 mM), osmotic (10 % PEG), cold (4 °C) and heat (42 °C) stresses, the highest transcript accumulation of gusA transcripts was observed in seedling roots between 24 and 72 h following the treatments (Fig. 6). Altogether, these results indicate that the 586-bp AlSAP promoter region has a behavior in rice, mirroring that of the AlSAP gene in its host, Aleuropus littoralis. These findings are in agreement with the presence of the predicted cis-

regulatory elements related to abiotic stress responses in the retained PrAlSAP promoter sequence (supplementary Table 2). The interesting feature of this promoter region is that despite its reduced size, its activity is stimulated by multiple stresses and localized in the same tissues, either with 586 bp (PrAlSAP) or after removing 83 bp of the 50 UTR (PrAlSAP–50 UTR). Wounding induces PrAlSAP promoter The in silico analysis of PrAlSAP revealed the presence of the cis-acting element induced by wounding (supplementary Table 2). To determine whether the AlSAP promoter is also induced by wounding, seeds and seedling leaves of the NB2.3 transgenic line and of the positive (CaMV–35S–gusA) or negative (WT) control lines were mechanically injured using a scalpel blade or a metal brush. Strong GUS activity was observed at wound sites in mature leaves (Fig. 7a) and in seed embryo and endosperm (Fig. 7b) of PrAlSAP: gusA plants, whereas leaf and seed tissues of PrCaMV35S–gusA plants exhibited similar deep blue staining whether the wound treatment had been applied or not (Fig. 7a, b). As expected, no GUS activity was detected in WT plants. The gusA transcript accumulation was monitored by northern blot analyses in wounded and unwounded leaf tissues of T2 rice plants harboring either the PrAlSAP–gusA or the PrCaMV35S–gusA construct (Fig. 7c). An increase of gusA transcripts was observed upon wounding in mature leaves of NB2.3 plants, whereas no obvious change of transcript accumulation was detected upon wounding in leaf samples of the PrCaMV35S–gusA control line (Fig. 7c). These results clearly demonstrate that the activity of the PrAlSAP promoter is stimulated by mechanical damage with a rapid response that results in elevated transcript level of the gene placed under its control. These results were confirmed by western blot (Fig. 7d).

Discussion The AlSAP gene and its ortholog in rice, OsSAP9, exhibit different regulations in response to abiotic stresses While AlSAP and OsSAP9 transcripts accumulate in root and leaves of young plants of Aeluropus littoralis and rice subjected to a range of abiotic stress treatment, they differ in their preferential tissue and stress-induced expression. In response to abiotic stresses, AlSAP transcripts accumulate at their highest level in roots, while OsSAP9 transcripts preferentially accumulate in leaves.

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Fig. 6 Relative expression level measured by real-time qPCR of gusA mRNA in roots and leaves of lines 2.3, 12.1 PrAlSAP–gusA rice plants under normal condition, and salt (150 mM NaCl), osmotic (10 % PEG8000), cold (4 °C) and heat (42 °C) stresses. Total RNA

was extracted from the rice seedlings at the four-leaf stage after various treatments. Amounts of cDNAs of rice were calibrated using the OsExp gene as an internal control. Results are presented as means and standard errors from three independent experiments

Also whereas AlSAP transcription is induced at a high level in response to all the applied stress treatments both in roots and leaves, OsSAP9 induction reaches its maximal magnitude following cold and heat stress in leaves. Our results of OsSAP9 expression pattern in rice are overall in concordance with a previous study conducted with different time courses and stress treatments and in mixed organs (Huang et al. 2008). It is worth noting that in the same study, overexpression of OsSAP9 in transgenic tobacco led to an enhanced tolerance to cold and thermal stresses, but to a parallel enhanced susceptibility to osmotic and salt stresses. This result contrasts with the overexpression of other OsSAP, (OsSAP1, OsSAP8 and OsSAP11 and AtSAP: AtSAP5) genes which generally conferred multiple stress tolerance in homologous or heterologous systems. This would point to a specialization of the OsSAP9 gene toward thermal stress response that is supported by our Q-PCR results. Vij and Tyagi (2006) detailed the genome-wide analysis of expression

of the AN1-type zinc finger protein family (SAP family in Vij’s article) in response to abiotic stress in indica rice. They found that all the members of the AN1-type zinc finger protein family are regulated by at least one type of stress, suggesting their involvement in abiotic stress response. Up-regulation by drought stress is notably observed in all the A20/AN1-type zinc finger protein genes, and OsSAP11 and OsSAP1 exhibited the most dramatic up-regulation. The in silico sequence analysis of the AlSAP promoter region reveals the presence of some abiotic and biotic stress-responsive cis-elements, transcription factor binding sites such as MYB, MYC, Dof and WRKY and several tissue-specific expression elements. The presence of these cis-elements related to various stresses is consistent with our previous finding showing that AlSAP is induced not only by various abiotic stresses (such as salt, osmotic heat and cold), but also by abscisic acid (ABA) and salicylic acid (SA) (Ben Saad et al. 2011).

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Fig. 7 Detection of GUS in line 2.3 PrAlSAP:gusA transgenic rice grains and leaves following wounding. Wounding induced GUS activity in leaves (a) and mature grain embryo or endosperm (b) (red arrows highlight wound sites). CaMV35S–gusA and WT rice plants were used as positive and negative controls, respectively. c Northern blot analysis was performed with gusA specific probe using the RNA isolated from the leaves of transgenic rice line 2.3 PrAlSAP–gusA,

CaMV35S–gusA and WT plants 1 h following the wound treatment compared to unwounded samples. Equal RNA loading in each lane was confirmed by ethidium bromide staining (lower panel). d Western blot analysis using the anti-GUS antibody to detect the presence of GUS protein in mature leaves. WT wild type, NW not wounded, W wounded; red arrows indicate the place of wounding

A 586-bp AlSAP promoter is sufficient for induction of gusA expression by multiple abiotic stresses in leaf and root tissues of rice

responsive elements in dehydration) and MYB recognition sites were present. It has been reported that the droughtinduced element DRE (dehydration responsive element) usually exists upstream of these drought-induced gene promoters, while ABA-induced gene promoters usually harbor an ABRE (Guiltinan et al. 1990; Mundy et al. 1990). However, other genes (e.g., RD22A) contain neither DRE nor ABRE elements, even if they were shown to be drought and ABA responsive. This third type of regulation has been reported to be mediated by the MYB and/or MYC recognition motifs that reside in the promoter region (Skriver and Mundy 1990). Transverse sections of organs of PrAlSAP:gusA lines subjected to histochemical staining revealed the presence of GUS reaction products in leaf phloem and mesophyll cells root exodermis, sclerenchyma, cortex, endodermis, pericycle and phloem cells. GUS staining of leaf blade and seminal roots of PrAlSAP:gusA seedlings was preferentially observed in the phloem. It is interesting that GUS activity is not observed in xylem, but only in phloem. Salinityresponsive genes have often a strong expression in vascular

Beyond the comparative relative expression of AlSAP and OsSAP9 genes, we were interested in the regulatory mechanisms controlling AlSAP gene expression in a heterologous system like rice. The real-time qPCR analysis of gusA mRNA levels in transgenic rice revealed that the reporter gene transcription under the control of PrAlSAP was highly induced by all the tested abiotic stresses. Accumulation of transcripts was more important in roots than in leaves. Further, in PrAlSAP:gusA plants grown under control conditions, GUS protein and GUS activity were detected at low level, while they increased considerably in plants subjected to dehydration, salinity, cold and heat treatments. This work clearly showed that, when used in rice, PrAlSAP reproduces the expression pattern of AlSAP in A. littoralis under stress or control condition. We did not find the drought-induced element DRE in the promoter region of AlSAP; however, the ABRE-like sequences (early

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bundles (Golldack et al. 2003; Shi et al. 2003). In reproductive organs, histochemical staining was observed in lemmas and paleas but not in stigma, filaments and anthers of young and mature flowers of the transgenic rice plants. Altogether, these results show that the tissue-specific and inducible features of the AlSAP promoter are maintained in rice, a C3 heterologous host. This is in contrast with the absence of GUSA activity and its protein as shown by western blot in roots of the dicot tobacco transformed with the same construct, even though there is a correct accumulation of gusA transcripts (Ben Saad et al. 2011). Promoter elements conferring specificity for one tissue have been studied extensively and are shown to be quite conserved (Benfey and Chua 1989; Kyozuka et al. 1993). However, it was demonstrated that in some cases even the specificity is conserved regulation will not work properly in heterologous system particularly with monocots elements in dicots or vice versa and the expression levels can be affected drastically (Benfey and Chua 1989; Gotor et al. 1993; Scha¨ffner and Sheen 1991). Correct accumulation of gusA transcripts observed in tobacco roots and the absence of GUS protein may suggest that a translational regulation of GUSA occurred in this dicotyledonous species that does not take place in monocots. It is clear from this study that AlSAP promoter has similar expression pattern in the C3 glycophyte monocot rice as in A. littoralis, a C4 halophyte monocot. Hence, it appears that monocots share conserved regulatory elements irrespective of whether they are C3 or C4 plants. The activity of the promoter region of SAP genes with promoter:gusA fusions has been previously investigated in Arabidopsis AtSAP5 (Kang et al. 2011) and At SAP10 (Dixit and Dhankher 2011) and rice OsSAP11 (also named OsDOG) (Liu et al. 2011). OsDOG is an atypical SAP gene, in that its major role lies in the regulation of GA homeostasis and negative maintenance of plant cell elongation (Liu et al. 2011). OsDOG promoter activity was rather constitutive in the plant. Our results therefore represent the first effort of functional analysis of a stressrelated monocot SAP gene in cereals. PrAlSAP is stimulated by wounding Histochemical assays in wounded leaf and seed tissues of PrAlSAP–gusA plants showed a clear staining at wound sites, suggesting that the AlSAP promoter is inducible by wounding in these tissues. This result was confirmed by both northern blot and western blot analyses. DNA sequence analysis showed that a wounding-related element, a W-box, resides in the delineated promoter region. The results of GUS activity are therefore consistent with the presence of this element. When plants are subjected to

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various kinds of stresses, such as wounding, they establish a strong defense system to protect themselves from further damage at stressed sites. W-boxes are well known to be involved in the induction of the wound and recognized by WRKY transcription factors (Nishiuchi et al. 2004; Rushton et al. 1996). The importance of W-boxes was illustrated recently by studies of the Arabidopsis transcriptome during systemic acquired resistance (Maleck et al. 2000). Though it is tempting to speculate that this W-box of the AlSAP promoter may confer some specificity of wound-induced expression, a more detailed functional analysis is required to identify the exact cis-elements responsible for wound response in the AlSAP promoter.

The AlSAP promoter is a good candidate for driving transgene expression in multiple stress-inducible and root-preferential manners Constitutive promoters, such as the CaMV35S promoter, have been widely used for engineering traits of interest in crop plants, because they conduct transgene expression in most plant tissues throughout plant growth and development and irrespective of environmental stimuli, taking advantage of limited temporal and spatial regulations. However, constitutively active promoters are not always desirable for sustainable expression of transgenes, also because constitutive overexpression of functional genes may result in homology-dependent gene silencing, particularly when the promoters are highly active (Vaucheret et al. 1998). It is therefore desirable to generate transgenic plants that accumulate transgenic products only under stimulated conditions. The results reported here provide a framework for pursuing the identification of regulatory elements in the AlSAP promoter. The specificity in high response to the wounding of this promoter might also be promising for an application in engineering biotic stress resistance traits into plants. Although the functions of SAP protein in plants are not clearly understood, the diversity of the sites of their expression and different stimuli used to activate them support the idea that the plant AlSAP proteins could be involved in many biological processes. In addition, we demonstrated that a short promoter region retains the tissue-specific and stress-induced expression pattern that is a useful feature for governing the expression of stress tolerance genes. It is also expected that this work will provide an important basis for further dissection of the regions that control the temporal- and spatial-specific expression and stress responses in the Aeluropus littoralis AlSAP gene. We do plan in the future to clone larger fragments of the AlSAP regulatory region as well as to conduct a functional dissection of the promoter.

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Author contribution statement EG and AH conceived and designed the research. RBS, WBR, DM, DMi, AA and JLV conducted the experiments. RBS, EG and AH analyzed the data and wrote the manuscript. All authors read and approved the manuscript. Acknowledgments The authors specially thank the National Program for Sciences, Technology, and Innovation (NPSTI, Project No. 11-Bio1828-02 2012–2014) in the Kingdom of Saudi Arabia for funding this work; the Visiting Professor Program of King Saud University Saudi Arabia; and the Agropolis Foundation under the REFUGE platform, CIRAD-Montpellier France for supporting this work. Compliance with ethical standards Conflict of interest of interest.

The authors declare that they have no conflict

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