Overexpression of a Medicago truncatula stress-associated protein gene (MtSAP1) leads to nitric oxide accumulation and confers osmotic and salt stress tolerance in transgenic tobacco

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Overexpression of a Medicago truncatula stress-associated protein gene (MtSAP1) leads to nitric oxide accumulation and confers osmotic and salt stress tolerance in transgenic tobacco

Aure´lie CharrierElisabeth Planchet Delphine CerveauChristine Gimeno-Gilles Isabelle Verdu Anis M. Limami Eric Lelie`vre

Received: 27 January 2012 / Accepted: 23 March 2012 ÓSpringer-Verlag 2012

Abstract The impact ofMedicago truncatulastress-asso- ciated protein gene (MtSAP1) overexpression has been investigated in Nicotiana tabacum transgenic seedlings.

Under optimal conditions, transgenic lines overexpressing MtSAP1revealed better plant development and higher chlo- rophyll content as compared to wild type seedlings. Interest- ingly, transgenic lines showed a stronger accumulation of nitric oxide (NO), a signaling molecule involved in growth and development processes. This NO production seemed to be partially nitrate reductase dependent. Due to the fact that NO has been also reported to play a role in tolerance acquisition of plants to abiotic stresses, the responses ofMtSAP1 overex- pressors to osmotic and salt stress have been studied. Com- pared to the wild type, transgenic lines were less affected in their growth and development. Moreover, NO content in MtSAP1overexpressors was always higher than that detected in wild seedlings under stress conditions. It seems that this better tolerance induced byMtSAP1overexpression could be associated with this higher NO production that would enable seedlings to reach a high protection level to prepare them to cope with abiotic stresses.

Keywords Abiotic stress NicotianaNitric oxide SeedlingStress-associated protein


cPTIO 2-(4-Carboxy-phenyl)-4,4,5,5-

tetramethylimidazoline-1-oxyl-3-oxide DAF-2DA 4,5-Diaminofluorescein diacetate MS Murashige and Skoog

NO Nitric oxide NR Nitrate reductase ROS Reactive oxygen species SAP Stress associated protein TLs Transgenic lines

WT Wild type


Plants are frequently exposed to different stresses that are induced by environmental changes. These disturbances can cause cellular dysfunctions which could lead to cell death (Knight and Knight 2001). In order to sense and to respond to unfavorable environmental situations, plants have developed complex cellular signaling mechanisms (Yamaguchi-Shinozaki and Shinozaki 2006; Nakashima et al.2009).

Stress-associated protein (SAP) families were charac- terized by the presence of A20/AN1 domains. The A20 zinc-finger (ZnF) domain was first identified in a TNF-a inducible protein in human cells and was characterized by multiple Cys2/Cys2 finger motifs. On the other hand, the AN1 domain has been identified in the C terminus of the ubiquitin-like protein coded by theXenopus Laevisanimal hemisphere 1 (AN1) maternal RNA (Linnen et al. 1993).

The AN1-type ZnF contains six conserved cysteines and two histidines that could potentially coordinate two zinc atoms (Linnen et al. 1993). The role of the A20/AN1 A. CharrierE. Planchet (&)D. Cerveau

C. Gimeno-GillesI. VerduA. M. Limami University of Angers, UMR 1345 Research Institute of Horticulture and Seeds (INRA, Agrocampus-Ouest, University of Angers), SFR 4207 Quasav, 2 Bd Lavoisier, 49045 Angers cedex, France

e-mail: elisabeth.planchet@univ-angers.fr E. Lelie`vre

University of Angers, UMR CNRS 6214 - UMR INSERM 1083, 2 rue Haute de Recule´e, 49045 Angers cedex, France

DOI 10.1007/s00425-012-1635-9


proteins has been well studied in animal immune systems (Huang et al.2004; Diatchenko et al.2005).

Recently, previous work highlighted the presence of A20/AN1 zinc-finger proteins across diverse organisms with a special emphasis on plants (Vij and Tyagi 2008).

The SAP gene family has been characterized in several reports to be involved in abiotic stress responses in plants (Mukhopadhyay et al.2004; Kanneganti and Gupta 2008;

Solanke et al. 2009; Stro¨her et al. 2009). Among these reports, it has been established that an OsiSAP8 gene product from rice (Oryza sativa) might act in the early phase of signal transduction pathway involved in stress responses (Kanneganti and Gupta 2008). However, SAP molecular functions remained unknown, although to date putative functions of A20/AN1 proteins had been pro- posed. Recently, AtSAP5 (Arabidopsis thaliana) was identified as possessing an E3 ubiquitin ligase activity (Kang et al. 2011). Moreover, it was reported that a receptor-like cytoplasmic kinase (OsRLCK253) interacted via A20 zinc-finger of SAP1/SAP11 and conferred abiotic stress tolerance in transgenic Arabidopsis plants (Giri et al.


Stress conditions induce modifications in many meta- bolic pathways. Nitric oxide (NO), a product of nitrogen metabolism, is known to be a key signaling molecule in plant signal transduction pathways (Neill et al. 2003;

Besson-Bard et al.2008). Several reports have shown the beneficial effects of NO on the physiological processes (seed germination, root organogenesis, senescence) (Leshem et al.1998; Lamattina et al.2003) and on the tolerance to biotic and abiotic stresses (Delledonne et al.1998; Zhao et al. 2004; Zhang et al. 2006). Among its actions, NO could interact with ROS (reactive oxygen species) which results from an oxidative component generated during all stresses (Delledonne et al.2001; Qiao and Fan 2008) and could regulate the gene expression involved in stress responses (Wendehenne et al.2004). Two major pathways for NO synthesis have been described, one reductive and one oxidative, involving nitrite and arginine, respectively, as substrates (Moreau et al.2010).

Previously, we issued a report on the cloning from a suppressive subtractive hybridization cDNA library of the first SAP in plant model legume, Medicago truncatula (Gimeno-Gilles et al.2011). The MtSAP1 protein sequence shared a strong homology with AtSAP7 and OsiSAP8 (54 and 62 %, respectively). Our results have revealed that MtSAP1 gene expression increased in the embryo during the acquisition of desiccation tolerance in M. truncatula.

Moreover, MtSAP1 protein accumulation was observed in response to different abiotic stresses in seeds and embryos (Gimeno-Gilles et al.2011).

To investigate the role of MtSAP1 in seedlings, and especially in response to abiotic stresses, MtSAP1

overexpression was performed in tobacco plants. Our results reveal that this overexpression seems to provide a high tolerance to osmotic and salt stress. This stress tol- erance could be related to high NO levels in tobacco transgenic lines. To our knowledge, this is the first inves- tigation which underlines a link between SAPoverexpres- sion and NO accumulation.

Materials and methods

Plant material and growth conditions

Experimentations were carried out on Nicotiana tabacum cv. Xanthi. In vitro, seeds (obtained from UMR PMS, University of Angers, France) were surface sterilised before sowing on solid half-strength Murashige and Skoog medium (MS 0.59) without sucrose and at pH 5.7 (Murashige and Skoog 1962). After stratification on MS 0.59 medium during 2 days at 4°C, seeds were placed under controlled growth conditions (25°C, 50 % relative humidity, 50–100 lmol m-2s-1 light intensity and 16 h light/8 h dark). These growth parameters previously described are considered as optimal conditions.

Obtention of transgenic tobacco overexpressing MtSAP1

Construction of MtSAP1 overexpression was obtained by cloning the complete coding sequence in the pMDC32 vector. Cloning was realized thanks to the GatewayÒ technology. Competent bacteria (EHA105 strain ofAgro- bacterium tumefaciens) were transformed with the pMDC32-MtSAP1construction. Four clones were pricked and tested by PCR using primers for pMDC32-MtSAP1 plasmid. Positive clones were selected for N. tabacum transformation. The Agrobacterium clones, containing a pMDC32-MtSAP1 construction, were placed in 2 mL of liquid LB medium supplemented with kanamycin (50 mg L-1) and rifampicin (25 mg L-1) at 30 °C and 100 rpm thus obtaining 0.3 DO (600 nm). The bacteria were again suspended in liquid M2a medium and used for explant infection. The infected tobacco leaves were placed in a Petri dish in darkness at 90 rpm and 22°C for 72 h.

Subsequently, they were washed in sterile water, placed on a selection medium containing 10, 20, 35 or 50 mg L-1of hygromycin, and transferred to a new medium approxi- mately every 10 days. When sufficiently developed buds were separated from calli and cultivated separately. Seed- lings were acclimatized when their roots were developed.

Later, for plant culture, seeds were sown directly on soil supplemented with perlite and placed in the same growth conditions.


Transgenic tobacco under abiotic stress conditions Wild type and T3 seeds of transgenic tobacco were sterilized with 70 % of ethanol for 30 s by inversion (Mukhopadhyay et al.2004). After removal of ethanol, seeds were immersed in a 2 % (v/v) sodium hypochlorite solution containing a drop of tween 20 for 5 min with a constant agitation. Then, seeds were washed six times in sterile water and dried on sterile whatman paper before sowing on different media.

Salt stress (NaCl; 125, 150 or 200 mM) and osmotic stress (D-mannitol; 80 or 300 mM) were performed in a solid MS 0.59 medium prior to autoclaving. The per- centage of germinated seeds (designated by the seed-coat rupture) was observed during 10 days, the other experi- ments were performed on 21-day-old seedlings.

RNA extraction and reverse transcription

For total RNA extraction, frozen seedlings were crushed in liquid nitrogen with a mortar and pestle. The isolation of RNA was realised with a Mini RNeasy plants mini-kit (Qiagen) in accordance to manufacturer’s instructions.

cDNAs were obtained by retrotranscription in an adequate buffer using 200 Units of M-MLV Reverse Transcriptase (Invitrogen), 2lg of random primers (Invitrogen) and 0.2 mM of dNTP (Promega) in the presence of 40 Units of a recombinant RNAsin ribonuclease inhibitor (Promega).

Reaction occurred during 1 h at 37°C in total volume of 50lL.

Real-time quantitative PCR

Reactions took place on the light cycler ABI Prism 7000 SDS (Applied Biosystems, Foster City, CA, USA). Every reaction was performed with a 3lL of a 1/10 (v/v) dilution of the first cDNA strands using an SYBR Green PCR Master Mix (Applied Biosystems), strictly following manufacturer’s instructions, with 200 nM of each primer in a total reaction of 25lL. Primers used for quantitative RT-PCR wereMtSAP1fw50-ACGTCAGTTGAAAACATC GTGAA-30; MtSAP1rv 50-TCGAGCATCAACAGCACT TG-30; NtNIA1fw 50-TGGATTGAACGCAACTTTTCC-30; NtNIA1rv 50-AACGGCGGTTCGGAGTTAA-30; NtNIA2fw 50-AACTCCGAACCACCGTTGAA-30 and NtNIA2rv 50-TGAAGTGGGACCGGTGTGA-30. Reaction occurred during a 2 min incubation at 50°C then 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 58°C.

The specificity of the PCR amplification procedure was checked with a heat-dissociation protocol (from 65 to 95°C) after the final cycle of PCR. Each measurement was carried out with at least two biological repeats, using a triplicate PCR reaction for determining Ct values. The ratio was calculated using an equation with normalization by a

reference gene (actin). Primers used wereNtActfw50-TCG CGAAAAGATGACTCAAATC-30 and NtActrev 50-CGGC TTGAATGGCGACATA-30.

MtSAP1 protein analysis by immunoblotting

Soluble proteins were extracted from frozen material in a 25 mM Tris–HCl buffer (pH 7.6) with 1 mM MgCl2, 1 mM EDTA and a cocktail of protease inhibitors (aprotinin 5 lg mL-1, leupeptin 2lg mL-1, pepstatin 0.1 lg mL-1, PMSF 1lM, Na3VO41 mM, NaF 5 mM). After denaturi- sation, equal amounts of protein (30 lg) were separated on an SDS-polyacrylamide gel (14 % (v/w) polyacrylamide).

Proteins were then transferred onto a PVDF membrane (Bio- Rad, Hercules, CA, USA).

After incubation with a rabbit polyclonal anti-SAP1 antibody (1/2,000) (Gimeno-Gilles et al. 2011), proteins were detected using a goat peroxidase-conjugated anti- rabbit antibody (1/4,000; Sigma) and visualized using ECL chemiluminescence (Bio-Rad).

Chlorophyll content

The total chlorophyll content from tobacco seedlings (20 mg FW) was calculated after extraction in N-N dime- thyl formamide (800lL; 2 h at RT) using the following formulae (Porra et al. 1989): [Chls a?b]=20.219 A645?8.029A663(A645andA663representing absorbance values). Chlorophyll content was expressed inlg mL-1. Endogenous NO detection by fluorescence

For fluorometric NO determination, the fluorophore 4,5- diaminofluorescein diacetate (DAF-2DA; Sigma) was used.

Root seedlings were pre-incubated with 10lM DAF-2DA for 30 min at 21°C in darkness and then rinsed twice with buffer (Mes; 5 mM, pH 6) to remove excess fluorophore.

DAF-2DA fluorescence (495 nm excitation and 515 nm emission wavelength) was observed by microscopy (LEICA DM4500B) connected to a fluorescence detection system L5. Fluorescence was expressed as arbitrary fluorescence units (A.U.) using an ImageJ software. For NO scavenging, root seedlings were incubated during 4 h with cPTIO (250 lM) before DAF treatment. For NR inactivation, (1) tungstate (250 lM), an NR inhibitor, was added to culture medium during 4 h before NO detection, or (2) seedlings were grown on MS 0.59medium in which nitrate has been replaced by NH4?(5 mM).

In vitro assay of NR activity

Following different treatments, seedlings were harvested, weighed and immediately quenched in liquid nitrogen. The


material was ground with liquid nitrogen and 2 mL of extraction buffer (100 mM Hepes pH 7.6, 10lM FAD, 15 mM MgCl2, 2 mM pefabloc, 0.5 % PVP, 0.5 % BSA) was added to 1 g FW. After continuous grinding until thawing the suspension was centrifuged (14,000g, 12 min, 4°C). After this centrifugation, aliquots of the extract were directly used for the colorimetric determination of nitrite content. With aliquots of the supernatant, the following assays were carried out:

1. Determination of NRact (?Mg2?): 200lL extract was added to 800 lL reaction mixture (100 mM Hepes–

KOH pH 7.6, 1 mM DTT, 10lM FAD, 15 mM MgCl2, 5 mM KNO3 and 0.2 mM NADH). After 5 min (24°C), the reaction was stopped by adding 125lL zinc acetate (0.5 M).

2. Determination of NRmax (?EDTA): 200 lL extract as above, but containing in addition 20 mM EDTA and 5 mM AMP (final concentrations) were preincubated at 25°C during 15 min. Afterwards, buffer (100 mM Hepes–KOH pH 7.6, 1 mM DTT, 10lM FAD and 15 mM EDTA) was added to a final volume of 1 mL.

The reaction was started by adding 5 mM KNO3and 0.2 mM NADH (final concentrations). Five minutes later, the reaction was stopped by adding 125lL zinc acetate (0.5 M). Following centrifugation (16,000g, 5 min), the supernatant was treated with 10lM phenazine methosulphate (PMS) to oxidize unreacted NADH (10 min in darkness), and the nitrite content was determined colorimetrically at 546 nm.

The activation state of NR was represented as the per- centage value of NRact (NRmax 100 %).

Statistical analysis of data

All data are presented as mean±SE of values from at least three independent experiments. A one-way ANOVA (analysis of variance) test was also performed. Different

letters are used to indicate means that differ significantly (P\0.05). For statistical tests, each transgenic line was only compared to wild type.


Overexpression ofMtSAP1gene in transgenic tobacco plants

To investigate the physiological function of MtSAP1, three independent transgenic tobacco lines overexpressing MtSAP1 under the control of CaMV 35S promoter were selected. The overexpression of the integrated MtSAP1 gene was determined by quantitative RT-PCR (Fig.1a). In comparison with wild type, only transgenic lines showed an MtSAP1 induction, proving the gene integration in tobacco genome and its expression. These results were confirmed by western blot analysis (Fig.1b).

Characterization of transgenic tobacco overexpressing MtSAP1in optimal growth conditions

To determine the impact of MtSAP1 overexpression on plant development, the seeds were sown on MS 0.59 medium and phenotypic analysis were realised on trans- genic tobacco lines. Firstly, it was showed that transgenic seedlings had a germination rate (84 % on average) lower than wild type (100 %) in optimal conditions after 7 days (Fig.2a), while the fresh weight measured on 21-day-old seedlings revealed an increase of biomass for the trans- genic lines with a gain of fresh weight above 2 mg per seedling compared to wild type (Fig.2b). After 28 days, this gain of fresh weight reached almost 10 mg per seed- ling for transgenic lines compared to wild type (data not shown). This allows one to presume a better development for the transgenic lines despite a lower germination rate, as shown on Table1. Root systems (number and length) and



0 1 2 3 4 5 6

cDNA copy number (106 )

WT Line 1 Line 2 Line 3


WT L1 L2 L3


Fig. 1 Characterization of tobacco transgenic lines overexpressing MtSAP1.aDetermination ofMtSAP1gene expression by quantitative real time RT-PCR. Each datum point is the mean of three independent

experiments±SE.bWestern blot on protein extracts from seedlings of wild type (WT) and transgenic lines (line 1,line 2and line 3).

MtSAP1 protein band is expected at thearrowlevel


vegetative parts (leaf number and diameter) of transgenic seedlings were more developed than wild type seedlings.

Moreover, chlorophyll content of transgenic lines was at least 1.5-fold higher than in the wild type line (Fig.2c).

The presence of these phenotypic differences between transgenic lines overexpressing MtSAP1 and wild type plants under optimal growth conditions indicated that expression ofMtSAP1could affect plant development and physiology.

Induction of NO accumulation inMtSAP1 overexpressing transgenic lines

NO is recognized as a signal molecule involved in many processes such as germination, plant development but also in response to abiotic stresses (Lamattina et al. 2003).

Experiments on NO levels and the potential involvement of

nitrate reductase in the NO biosynthetic pathway have been conducted on transgenic lines overexpressingMtSAP1.

Detection of endogenous NO in the roots of transgenic seedlings under optimal conditions has been achieved through the method using DAF-2DA fluorescence micros- copy. NO fluorescence was clearly detected in transgenic lines compared to wild type (Fig.3a), suggesting that MtSAP1overexpressing seedlings produced more NO than wild type. Estimation of the NO levels, observed in the photographs, confirmed that NO accumulation of trans- genic lines was 2 to 2.3 times higher than in wild type (Fig.3b). As negative control, the NO scavenger cPTIO reduced strongly NO fluorescence, showing the specificity of NO detection.

In order to identify whether NR could be involved as a NO potential source, root seedlings were incubated with tungstate, molecule widely used in plant NO research as

0 2 4 6 8

0 20 40 60 80 100

Germination rate (%)

Time (days)

WT Line 1 Line 2 Line 3

a b


0 7 8 9 10 11 12

Fresh weight (mg seedling-1 )

WT Line1 Line 2 Line 3

Chlorophyll content (µg mg-1 FW)

a b

b b

b b b

a WT Line 1 Line 2 Line 3

WT Line 1 Line 2 Line 3 0.4


0.0 0.6

a b

b b

b b b


Fig. 2 Effects ofMtSAP1 overexpression on seed germination and plant

phenotype in transgenic tobacco lines.aGermination rate was observed during 7 days. Fresh weight (b) and chlorophyll content (c) were determined in WT and in three transgenic lines growing on MS 0.59medium during 21 days. Each value represents the mean±SE of three independent experiments.

Different lettersare used to indicate means that differ significantly (P\0.05) according to a one-way ANOVA test. For statistical tests, transgenic lines were only compared to wild type

Table 1 Comparison of various growth parameters of WT and tobacco transgenic lines grown under optimal conditions

Each value represents the mean of three independents samples.

SE is indicated in brackets

Lines Length primary root (cm)

Length secondary root (cm)

Secondary root number

Leaf number Leaf diameter WT 1.70 (±0.17) 0.40 (±0.40) 0.50 (±0.50) 5.67 (±0.44) 0.35 (±0.01) Line 1 2.76 (±0.34) 1.23 (±0.03) 1.19 (±0.23) 6.57 (±0.38) 0.54 (±0.05) Line 2 3.92 (±0.55) 2.53 (±0.08) 2.27 (±0.22) 7.77 (±0.84) 0.71 (±0.08) Line 3 2.70 (±0.10) 1.20 (±0.02) 2.00 (±0.00) 6.00 (±0.00) 0.60 (±0.02)


NR inhibitor (Planchet et al.2005; Cantrel et al.2011). The basic NO level observed in wild type roots under optimal conditions was slightly reduced in the presence of tungstate instead of molybdenum (Fig.4a). Otherwise, the loss of NO fluorescence varied from 36 to 48 % following NR inhibitor treatment in transgenic lines (Fig.4a). Consistent with these observations, roots from ammonium fed (nitrate free) transgenic lines showed also a reduction in NO pro- duction (Fig.4a).

Moreover, the expression profiles of two NR genes in Nicotiana tabacum (Nia1 and Nia2) were studied by quantitative RT-PCR (Fig.4b). Nia1 expression in MtSAP1-overexpressing lines was induced 3.2 to 6.2 times more than wild type, whereasNia2 expression seemed to be slightly affected byMtSAP1overexpression. Otherwise, while the NR activation state was 33.6 % in wild type, this rate increased to 43.4 %, for transgenic lines (Fig.4b inset).

Osmotic and salt stress tolerance acquisition by overexpression ofMtSAP1

To evaluate the impact of MtSAP1 overexpression on osmotic stress tolerance, germination rate and seedling growth were observed under osmotic stress conditions induced byD-mannitol (300 mM). The results revealed that the germination rate was lower in wild type line (61.2 %) than in transgenic lines (higher than 78.5 %) (Fig.5a). This data seemed to show a better tolerance to osmotic stress in transgenic lines, which appeared to be correlated with a less marked decrease in fresh weight (52.2 against 75.1 % for WT) (Fig.5b) and a greater development (Fig.5c).

In the same way, salt stress tolerance has been also tested. In general, the germination rate (Fig.6a), the fresh weight values (Fig.6b) and the development analysis (Fig.6c) indicated that wild type under salt stress condi- tions was significantly affected in comparison with

WT Line 1 Line 2 Line 3

0 10 20 30 40 50 60

Fluorescence intensity (A.U.)





Line 2 Line 3

Line 1 WT


Fig. 3 NO detection in tobacco transgenic lines overexpressing MtSAP1.aVisualization of in vivo NO production was observed in root seedlings (21 old days after post imbibition) growing on MS

0.59medium±cPTIO (250lM). Pictures are representative data.

b Fluorescence quantification was expressed as ratio showing each line compared to WT under optimal conditions (n=4±SE)


transgenic lines. Indeed, wild type seedlings, under salt stress condition, stopped their development (producing less leaves and roots), but also showed a chlorosis apparition.

Quantification of total chlorophyll revealed that wild type lost chlorophyll (49 %) while transgenic lines seemed to produce more chlorophyll (19, 55 and 37 % for the three lines, respectively) under salt stress (Fig.6d).

NO production under osmotic and salt stress in wild type and transgenic lines

Under osmotic and salt stress, NO production was strongly stimulated in stressed wild type, but transgenic lines overexpressing MtSAP1 accumulated always more NO

than wild type seedlings (Fig.7a). It is to be noted that this NO production in transgenic lines seemed to remain stable in comparison to optimal conditions. Both stress treatments induced an increase of NR gene expression in wild type compared to optimal conditions, except forNIA2under salt stress condition (Fig. 7b, c). Interestingly, it should noted that the expression level of NR genes was generally higher in transgenic lines under osmotic and salt stress compared to stressed wild type (Fig.7b, c). These data appeared to be correlated with previous results in which NO production in wild type, whatever the culture conditions, remained lower compared to transgenic lines. Although the NR activation state under optimal conditions showed a trend to be higher in lines overexpressing MtSAP1 than in wild type, no

a b

Nia 2 Nia 1

0 2 4 6 8

Fold induction (ratio lines vs WT)

WT Line 1 Line 2 Line 3

20 30 40 50 60

NR activation state (%)


WT Line 1 Line 2 Line 3 0

10 20 30 40 50

60 MS

Tungstate NH4Cl

Fluorescence intensity (A.U.)

a a

a b



a b

b b

Fig. 4 NO production and NR involvement in tobacco transgenic lines overexpressingMtSAP1under normal conditions.a NO quan- tification in seedlings treated with an NR inhibitor (tungstate;

250lM) or grown on NH4? medium.bExpression profiles of NR genes and (binset) NR activation state in seedlings under optimal conditions. TLs represents the mean value of transgenic lines. Fold

inductions ofNiagenes for each line were compared to WT under optimal conditions. Each value represents the mean±SE of three independent experiments.Different lettersare used to indicate means that differ significantly (P\0.05) according to a one-way ANOVA test. Statistical tests forNIA1andNIA2genes have been performed independently

0 2 4 6 8 10

0 20 40 60 80 100

Germination rate (%)

Time (days)

WT Line 1 Line 2 Line 3



b WT Line 1 Line 2 Line 3

80 70 60 50 40 0

Loss of fresh weight (%) (stress vs control conditions)


b b b

WT Line 1 Line 2 Line 3

Fig. 5 Effects ofMtSAP1 overexpression on seed germination and plant

phenotype in transgenic tobacco lines under osmotic stress conditions. Germination rate (a) and decrease in fresh weight (b) were measured after

D-mannitol treatment (300 mM).cSeedling photograph was taken after osmotic treatment (21 days).

The photograph is a representative datum. Each value represents the mean±SE of three independent

experiments.Different letters are used to indicate significant difference (P\0.05) according to a one-way ANOVA test


significant differences for NR activation state could be observed under stress treatments between wild type and transgenic lines (Fig.7d). Otherwise, it could be observed that the NR activation state for wild type seedlings seemed to increase under D-mannitol and NaCl treatments (48.4 and 59 %, respectively) compared to optimal conditions (33.6 %; Fig.7b).


In the present work, it was established thatMtSAP1over- expression in N. tabacum induced morphological and physiological changes in seedlings under optimal condi- tions, i.e. biomass increase, better development of root and vegetative systems and higher chlorophyll content. Inter- estingly, our results have also revealed that MtSAP1 overexpression provoked a stronger NO accumulation in comparison with wild type. In our knowledge, this study reports for the first time a link between A20/AN1 proteins and NO production. Many previous reports, describing the NO involvement in developmental processes, led us to think that phenotypical characteristics observed in trans- genic lines could be related to their higher NO production.

Indeed, available evidence in many species showed that NO promoted adventitious root formation (Lanteri et al.

2006) and lateral root initiation (Correa-Aragunde et al.

2004; Zandonadi et al. 2010) and induced growth

elongation in roots (Gouvea et al.1997) and leaf expansion (Leshem1996; An et al.2005).

The source of NO production in higher plants is sub- jected to debate and is predicted to be mediated by NR, nitrite-dependent mitochondrial electron transport and/or NOA (NO associated protein) (Rockel et al. 2002;

Planchet et al. 2005; Moreau et al. 2010). However, the NR-dependent NO formation is the only NO source in plants that is well established, and for which genes and enzymes and their regulation have been intensively inves- tigated (Kaiser et al. 2011). In the present study, we have highlighted a higher NR gene induction (NIA1andNIA2) in transgenic lines than wild type under optimal conditions.

These results led to think that NR could participate in NO production in N. tabacum seedlings overexpressing MtSAP1. Consequently, MtSAP1 overexpression may induce some nitrogen metabolism modifications and could permit to increase nitrate assimilation that promotes an optimal plant development.

In addition to NO participation in physiological and developmental functions in plants, this key molecule has been suggested to be involved in responses to environ- mental stresses such as drought, salt, heat, cold stress or diseases (Hong et al. 2004; Qiao and Fan 2008). In the present work, the physiological parameters observed under osmotic and salt stress conditions showed that transgenic lines overexpressing MtSAP1 presented a better growth (germination rate, biomass and development) than wild type. Previous studies have already established the

Chlorophyll content (ratio lines vs MS)

0 2 4 6 8 10

0 20 40 60 80 100

Germination rate (%)

Time (days)

WT Line 1 Line 2 Line 3

a b

c d

WT Line 1 Line 2 Line 3

80 60 40 20 0

Loss of fresh weight (%) (stress vs control conditions)

WT Line 1 Line 2 Line 3

WT Line 1 Line 2 Line 3 a

b b


a b

b b

WT Line 1 Line 2 Line 3

WT WT Line 1 Line 2 Line 3

WT Line 1 Line 2 Line 3







b b

Fig. 6 Effects ofMtSAP1 overexpression on seed germination and plant

phenotype in transgenic tobacco lines under salt stress

conditions. Germination rate (a) and decrease in fresh weight (b) were measured after NaCl treatment (200 mM). Seedling photograph (c) and chlorophyll content determination (d) (expressed as a ratio comparing each stressed line vs.

itself under optimal conditions) were made after salt treatment (21 days; 150 mM). Means are from three independent experiments (±SE).Different lettersare used to indicate significantly difference (P\0.05) according to a one- way ANOVA test. The photograph is a representative datum


induction of SAP gene in response to various stresses (Vij and Tyagi 2006) and their involvement in tolerance acquisition (Solanke et al.2009; Ben Saad et al.2010). For example,OsiSAP8overexpression in rice transgenic lines conferred to plants a better development under salt, drought and cold stress (Kanneganti and Gupta2008). In our study, we hypothesize that osmotic and salt stress tolerance of MtSAP1-overexpressing lines could be related to their higher NO levels observed previously under optimal con- ditions. Indeed, this assumption was supported by the fact that transgenic lines under treatments showed a better development and an increase of chlorophyll content. Pre- vious works have already established the NO involvement in an iron remobilization process leading to a better pho- tosynthetic capacity and chlorophyll synthesis in leaves (Graziano et al. 2002), but also that NO occured in the protection of chloroplast membranes against ROS gener- ated by all types of biotic or abiotic stresses (Laxalt et al.

1997). Moreover, the NO implication was also demon- strated in salt stress tolerance acquisition through its role in the Na? sequestration by increasing activities of proton- pump and Na?/H? antiport (Zhang et al. 2006). Effec- tively, in the present study, wild type lines showed an increase of NO production in response to osmotic and salt treatments. This NO accumulation was partially correlated to NR gene induction (Nia1 andNia2) and to a plausible increase of NR activation state under stress. According to this, previous works have shown a NR-mediated NO accumulation in response to osmotic stress (Kolbert et al.

2010), salt stress (Liu et al. 2007) and ABA treatment

(Desikan et al.2002). Very recently, SIZ1, a regulator of signalling pathways that mediate responses to abiotic stresses and nutrient deficiency (Miura et al. 2005; Catala et al. 2007), has been proved to regulate nitrogen metab- olism through its E3 SUMO ligase function inArabidopsis thaliana (Park et al. 2011). This sumoylation by AtSIZ1 has been shown to stimulate NR activity and increase NO production (Park et al.2011). Interestingly,AtSAP5, which encodes a protein with both A20 and AN1 zinc finger motifs, acts through its E3 ubiquitin ligase activity as a positive regulator of stress responses in Arabidopsis (Kang et al. 2011). Thus, the fact that MtSAP1 exhibits A20 and AN1 zinc finger motifs, we can cautiously hypothesize that MtSAP1 could induce an E3 ligase activity dependent NO production.

Even if it is very interesting to note that MtSAP1- overexpressing seedlings did not seem to produce more NO under treatments compared to optimal conditions, NO levels in transgenic lines remained always higher than the wild type lines. This may be explained by a regulation of NO turn-over that could permit to avoid cytotoxic NO effects. Indeed, it has been reported that NO, at too high concentrations, could injure nucleic acids, proteins and membrane integrity, but also could reduce photosynthesis and respiration (Qiao and Fan 2008). The constant NO production induced by the MtSAP1 overexpression could permit the plant prepara- tion to cope with stress quickly and to acquire a higher tolerance threshold. In fact, transgenic lines may have reached a high protection level against osmotic and salt

a b



0 20 40 60 80

NR Activation state (%)

MS D-mannitol NaCl WT Line 1 Line 2 Line 3

0 20 40 60 80

Fluorescence intensity (A.U.) MS

D-mannitol NaCl

2 4 6 8 10 12 14

Fold induction of NIA1 gene (vs unstressed WT)

MS D-mannitol NaCl

MS D-mannitol NaCl

Fold induction of NIA2 gene (vs unstressed WT) 3.0 d

2.5 2.0 1.5 1.0 0.5 0.0

WT Line 1 Line 2 Line 3

WT Line 1 Line 2 Line 3

Fig. 7 NO production and NR expression under stress conditions in transgenic tobacco lines.aNO quantification in root seedlings submitted to osmotic (D-mannitol; 80 mM) and salt (NaCl; 125 mM) stress during 6 h. Effects of osmotic and salt stress onNIA1(b) and NIA2(c) gene expressions.

dNR activation state under osmotic and salt stress.TLs represent the mean value of transgenic lines. Each datum point is the mean of three independent experiments±SE


stress through the NO accumulation. The NO actions could be due to its antioxidant functions (modulation of O2-

formation and inhibition of lipid peroxidation) (Neill et al.2003) and/or its abilities to induce responsive genes (Parani et al. 2004).

In conclusion, we showed that the combination of MtSAP1 and NO could play an important role in tolerance acquisition to osmotic and salt stress. The molecular mechanisms induced by this SAP protein family were poorly understood, but this study and previous reports could provide some lines of work. The localisation in the nucleus of other SAP proteins (Dixit and Dhankher2011) and the MtSAP1 conformation may suggest that MtSAP1 could play a role of transcription factor. This function could lead to upregulate genes coding for proteins involved in tolerance processes such as cell protection and NO pathways.

Acknowledgments The authors are grateful to Mr Michael Jones for English language correction. Funding was provided by QUALI- SEM contract with Region Pays de la Loire, France. The postdoctoral fellowship of Aure´lie Charrier is supported by a grant from Angers University.


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