Polyamines contribute to salinity tolerance in the symbiosis Medicago truncatula-Sinorhizobium meliloti by preventing oxidative damage

Accepted Manuscript Polyamines contribute to salinity tolerance in the symbiosis Medicago truncatulaSinorhizobium meliloti by preventing oxidative damage Miguel López-Gómez, Javier Hidalgo-Castellanos, J. Rubén Muñoz-Sánchez, Agustín J. Marín-Peña, Carmen Lluch, José A. Herrera-Cervera PII:

S0981-9428(17)30146-8

DOI:

10.1016/j.plaphy.2017.04.024

Reference:

PLAPHY 4870

To appear in:

Plant Physiology and Biochemistry

Received Date: 24 January 2017 Revised Date:

24 April 2017

Accepted Date: 25 April 2017

Please cite this article as: M. López-Gómez, J. Hidalgo-Castellanos, J.Rubé. Muñoz-Sánchez, Agustí.J. Marín-Peña, C. Lluch, José.A. Herrera-Cervera, Polyamines contribute to salinity tolerance in the symbiosis Medicago truncatula-Sinorhizobium meliloti by preventing oxidative damage, Plant Physiology et Biochemistry (2017), doi: 10.1016/j.plaphy.2017.04.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title

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Polyamines contribute to salinity tolerance in the symbiosis Medicago truncatula-

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Sinorhizobium meliloti by preventing oxidative damage.

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Authors

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Miguel López-Gómez, Javier Hidalgo-Castellanos, J. Rubén Muñoz-Sánchez,

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Agustín J. Marín-Peña, Carmen Lluch, José A. Herrera-Cervera

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Departamento de Fisiología Vegetal, Facultad de Ciencias, Universidad de

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Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain.

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Corresponding Author

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Miguel López-Gómez

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e-mail: [email protected]

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Departamento de Fisiología Vegetal, Facultad de Ciencias, Universidad de

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Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain

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ACCEPTED MANUSCRIPT Abstract

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Polyamines (PAs) such as spermidine (Spd) and spermine (Spm) are small ubiquitous

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polycationic compounds that contribute to plant adaptation to salt stress. The positive

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effect of PAs has been associated to a cross-talk with other anti-stress hormones such as

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brassinoesteroids (BRs), also involved in anti-stress responses in plants. In this work we

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have studied the effects of exogenous Spd and Spm pre-treatments in the response to

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salt stress of the symbiotic interaction between Medicago truncatula and Sinorhizobium

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meliloti by analyzing parameters related to nitrogen fixation, oxidative damage and

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cross-talk with BRs in the response to salinity.

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Exogenous PAs treatments incremented the foliar and nodular Spd and Spm content

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which correlated with an increment of the nodule biomass and nitrogenase activity.

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Exogenous Spm treatment partially prevented proline accumulation which suggests that

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this polyamine could replace the role of this amino acid in the salt stress response.

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Additionally, Spd and Spm pre-treatments reduced the levels of H2O2 and lipid

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peroxidation under salt stress. PAs induced the expression of genes involved in the BRs

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biosynthesis which support a cross-talk between PAs and BRs in the salt stress response

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of the M. truncatula-S. meliloti symbiosis

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In conclusion, exogenous PAs improved the response to salinity of the M. truncatula-S.

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meliloti symbiosis by reducing the oxidative damage induced under salt stress

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conditions. In addition, in this work we provide evidences of the cross-talk between PAs

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and BRs in the adaptive responses to salinity.

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Keywords

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Polyamines, brassinosteroids, salt stress, symbiosis, Medicago truncatula.

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Abbreviations

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PAs, polyamines; BRs, brassinosteroids; Put, putrescine; Spd, spermidine; Spm,

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spermine; Cad, cadaverine; Homspd, homospermidine; MDA, malondialdehyde; Pro,

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proline; NFR, nitrogen fixation rate; NFW, nodule fresh weight.

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1. Introduction The diamine putrescine (Put) and the polyamines (PAs) spermidine (Spd) and spermine

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(Spm) are small ubiquitous polycations involved in numerous processes in all living

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organisms [1]. In plants, PAs are involved in cell division, embryogenesis, senescence,

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floral development and fruit ripening [2]. In addition, PAs play an important role in the

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response of plants to adverse environmental conditions due to their polycationic nature

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[3] [4]. Among these adverse conditions, soil salinity is one of the most important

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abiotic factors limiting crop productivity all over the world, especially in arid and

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semiarid regions, and is predicted to get worse under climate change conditions [5]. Salt

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stress adversely affects plant development and productivity by generating ion toxicity,

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osmotic stress, water deficits and oxidative damage through the production of reactive

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oxygen species (ROS) including hydrogen peroxide (H2O2) among others [6].

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Legumes are classified as salt-sensitive crop species and their productivity is

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particularly affected by soil salinity because nodular nitrogenase activity, responsible

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for the nitrogen supply to the plant in symbiosis with soil bacteria known as rhizobia,

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markedly decreases upon exposure to saline conditions [7] [8]. Root nodules of legumes

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have been found to contain a high variety and concentration of PAs, some of them of

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bacteroidal origin [9], however, little is known about the role of PAs within the root

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nodules. A cross-talk between PAs and other plant growth regulators such as

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brassinosteroids (BRs), has been described in the response to abiotic stresses [10], [11],

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[12]. BRs biosynthesis is regulated at the transcriptional level of the biosynthetic genes

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by other plant hormones such as auxin [13] and in addition, BRs have been shown to be

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involved in the systemic regulation of the root nodule formation in soybean [14] by a

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modification of the PAs levels in leaves [15].

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ACCEPTED MANUSCRIPT Exogenous addition of PAs has been extensively used as a strategy to enhance tolerance

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to salinity. There is a large body of evidence suggesting that exogenous application of

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PAs could preserve plant cell membrane integrity, minimize growth inhibition caused

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by stress, reduce superoxide radical and H2O2 contents and increase activities of

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antioxidant enzymes (reviewed by [2]).

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In this work we have studied the effects of exogenous Spd and Spm in the adaptation to

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salt stress of the symbiosis M. truncatula-S.meliloti by analyzing parameters related to

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nitrogen fixation, oxidative damage and cross-talk with BRs in the response to salinity.

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2. Material and Methods

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2.1 Biological material and growth conditions

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Medicago truncatula (var. Jemalong) seeds were scarified by immersion in concentrated

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H2SO4 for 5 min, washed with sterile water, surface sterilized by immersion in NaClO

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50% (v/v) plus Tween-20 for 10 min and germinated onto 1.0% water-agar plates at 25

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ºC in the darkness. After 3 days, Medicago seedlings were transferred to sterile

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vermiculite:perlite (3:1) and watered with a modified nitrogen free [16] nutrient

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solution. Two days later, M. truncatula seedlings were inoculated with S. meliloti 1021

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strain (c. 109 cell ml-1) grown in a TY medium.Plants, in individual pots of about 200

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ml, were grown in a controlled environmental chamber with a 16/8 h light-dark cycle,

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23/18ºC day night temperature, relative humidity 55/65% and photosynthetic photon

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flux density (400-700 nm) of 450 µmol m-2 s-1 supplied by combined fluorescent and

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incandescent lamps. Six weeks after sowing plants were subjected to Spd and Spm

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treatments by the addition of 0.1 mM of each polyamine to the nutrient solution.

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Control plants were watered with the same nutrient solution without polyamines. Salt

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ACCEPTED MANUSCRIPT treatment was applied two weeks later by the addition of 75 mM NaCl to the watering

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nutrient solution. Control plants were watered with a NaCl-free nutrient solution. Plants

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were harvested 10 weeks after sowing with polyamines and NaCl treatments lasting 4

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and 2 weeks, respectively. Nodules and leaves were frozen at -80 ºC for further

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analyses. Samples of leaves, stems and roots were dried at 70 ºC for 24 h and their dry

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weight determined.

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2.2 Nitrogen fixation

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Nitrogenase activity (E.C. 1.7.9.92) was measured as the representative H2-evolution in

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an open-flow system [17] using an electrochemical H2 sensor (Qubit System Inc.,

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Canada). H2 production was recorded in intact nodulated roots of plants. Apparent

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nitrogenase activity (ANA, rate of H2 production in air) was determined under N2:O2

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(80%:20%) with a total flow of 0.4 l min-1. After reaching steady-state conditions total

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nitrogenase activity (TNA) was determined under Ar:O2 (79%:21%). Nitrogen fixation

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rate (NFR) was calculated as (TNA-ANA)/3. Standards of high purity H2 were used to

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calibrate the detector.

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2.3 Determination of free polyamines in leaves and nodules

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Nodule and leaves extracts were prepared from 0.2 g of fresh tissue with 0.6 ml of 5%

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(v/v) cold perchloric acid (PCA) and incubated 24 h at 4 ºC. The homogenate was

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centrifuged (3,000xg, 5 min, 4 ºC) and 0.2 ml aliquots of the supernatant were

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dansylated as described below. The analysis of free PAs was performed with HPLC

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(Agilent Technologies 1260) equipped with a reverse phase column (4.6 x 250 mm

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ACCEPTED MANUSCRIPT C18) after derivatization with dansyl chloride (Sigma). Derivatization was performed by

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mixing 0.2 ml aliquots of the extracts prepared as described above with 0.4 ml of dansyl

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chloride (prepared fresh in acetone, 10 mg/ ml) and 0.2 ml of saturated sodium

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carbonate. After brief vortexing, the mixture was incubated in darkness at room

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temperature overnight. Excess dansyl reagent was removed by reaction with 0.1 ml (100

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mg/ml) of added proline, and incubation for 30 min. Dansylpolyamines were extracted

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in 0.5 ml toluene. The organic phase was collected and evaporated to dryness under a

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stream of nitrogen, and redissolved in 0.1 ml acetonitrile.

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Column flow was 1.5 ml min-1 and the elution gradient was prepared with eluent A

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(water) and eluent B (acetonitrile). The column was equilibrated with 70% B and 30%

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A before injecting 0.01 ml samples. This was followed by a linear gradient ending with

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100% B after 9 min. The final step was held for 4 min before regenerating the column.

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Detection was done with a fluorometer using excitation and emission wavelengths of

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415 and 510 nm, respectively, according to [18]. A relative calibration procedure was

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used to determine the PAs in the samples, using 1,7- diaminoheptane (HTD) as internal

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standard and PAs standards amounts ranging from 0.3 to 1.5 nmol purchased from

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Sigma. Results were expressed as nmol g-1 fresh weight.

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2.4 Proline determination

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Proline was determined by the ninhydrine method [19]. Briefly, 250 mg of plant

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material was homogenized in 3 ml of 95% (v/v) ethanol at room temperature and

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centrifuged at 2,000 x g for 5 min. 300 µl of distilled water and 2 ml of ninhydrine

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reagent were added to an aliquot of 200 µl of extract. The mixture was boiled for 60

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min, and the reaction was stopped in an ice bath. The chromophore obtained was 7

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extracted with 6 ml of toluene by vigorous shaking for 20 s. Absorbance of the resulting

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organic layer was measured at 520 nm with Varian UV-VIS spectrophotometer.

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Calibration was made using L-Pro as a standard.

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2.5 Lipid peroxidation

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Lipid peroxidation was measured by the level of malondialdehyde (MDA), a product of

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lipid peroxidation, using a reaction with thiobarbituric acid (TBA) as described by [20].

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Fresh samples (100 mg) were ground in a mixture of 1 ml trichloroacetic acid (TCA)

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(20% w/v) and 0.2 ml of 4% (w/v) butylatedhidroxytoluene in ethanol, at 4 ºC. After

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centrifugation (10,000 x g for 15 min), 0.25 ml aliquots of the supernatant were mixed

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with 0.75 ml of 0.5% (w/v) thiobarbituric acid in 20% TCA and the mixture was

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incubated at 94 ºC for 30 min. The reaction was stopped by cooling in an ice bath for 15

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min. Reaction tubes were centrifuged at 10,000 x g for 15 min and supernatants were

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used to determine the absorbance at 532 nm. The value for non-specific absorption at

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600 nm was subtracted.

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2.6 Histochemical and quantitative detection of H2O2 in leaves

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The histochemical analysis of H2O2 was performed in leaves of two weeks old plants

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grown axenically in glass test tubes. Plants were pretreated with Spm 0.1 mM 24 h

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before treatment with NaCl to a final concentration of 150 mM for 24h. The H2O2 was

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localized histochemically by staining leaves with 1% 3,3-diaminobenzidine (DAB)

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solution. Leaves were immersed in such solution until brown spots appeared due to the

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boiling ethanol to see the spots.

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Quantitative analysis of H2O2 was performed spectrophotometrically after reaction with

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KI [22]. The reaction mixture consisted of 0.5 ml 0.1% trichloroacetic acid (TCA) leaf

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extract supernatant, 0.5 mL of 100 mM K-phosphate buffer and 2 mL reagent (1 M KI

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w/v in fresh double-distilled water H2O). The blank probe consisted of 0.1% TCA in the

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absence of leaf extract. The reaction was developed for 1 h in darkness and absorbance

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measured at 390 nm. The amount of H2O2 was calculated using a standard curve

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prepared with known concentrations of H2O2.

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2.7 Gene expression analysis in leaves

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For gene expression analysis, plants were grown axenically onto filter paper in glass test

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tubes containing 10 ml of nutrient solution [23]. Two weeks after sowing, plants were

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pre-treated with Spd and Spm to a final concentration of 0.1 mM for 24h and then, NaCl

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to a final concentration of 150 mM was added. After 48h and 24h of PAs and NaCl

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treatments, respectively, plants were harvested and frozen at -80 ºC. A total of 6 plants

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per repetition and treatment were used for RNA preparation.

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Total RNA was extracted using an RNeasy plant mini kit (Macherey-Nagel, Germany)

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followed by treatment with DNaseI (Ambion, Texas, USA) for genomic DNA removal.

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First-strand cDNA was reverse transcribed from 1.5 µg of DNase-treated total RNA. All

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DNase-treated total RNA samples were denatured at 65 ºC for 5 min followed by quick

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chill on ice in 12 µl of reaction mixture containing 0.5 µg oligo-dT adapter primers and

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1 µl of 10 Mm deoxy-nucleotide triphosphate.

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inhibitor and 2 µl of 0.1 M dithiothreitol (DTT), the reaction was preheated at 37 ºC for

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3 min before the addition of 1 µl (200 U) of iScript reverse transcriptase (Bio-Rad,

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California, USA). The reaction mixture was incubated at 42 ºC for 50 min, followed by

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heat inactivation at 70 ºC for 15 min. The resulted first strand cDNA was amplified

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using gene specific primers (Table 1) designed from the transcribed region of each gene

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by using the Clone Manager Professional Suite software. The actin gene was used as

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loading control.

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PCR amplifications were performed in 20 µl reactions mixture as follows: one cycle of

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2 min at 94 ºC, 35 cycles of 30 s at 94 ºC, 30 s at 56 ºC and 1 min at 72 ºC and a final

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extension at 72 ºC for 10 min. PCR products were electrophoretically separated in 1%

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(w/v) agarose gels. Real-time PCR (qPCR) was performed using a SYBR Green PCR

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Master kit containing the Platinum Taq DNA polymerase (Invitrogen) on an iCycler IQ

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thermocycler (Bio-Rad). PCR amplification mixtures contained 10 ng of template

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cDNA and 0.5 U of polymerase. The cycling conditions were chosen according to the

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manufacturer. They comprised 10 min polymerase activation at 95 ºC and 35 cycles at

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95 ºC for 15 s, 55 ºC for 30 s and 72 ºC for 20 s. Results were quantified with the DDCt

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method [24]. Transcript levels were normalised to actin gene expression. The primer

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sequences for qPCR analysis are provided in Table 1.

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2.8 Statistical analysis

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Experiments were arranged in a completely randomized design with totally 60 plants

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per treatment. The data were subjected to an analysis of two-way ANOVA with

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exogenous polyamines as one factor and salt treatment as the other using the 10

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significant differences between treatments were determined by LSD (P ≤ 0.05).

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Experiments were performed three times in triplicate. Mean values ± SE error bars are

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represented

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3. Results

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3.1 Exogenous PAs induce plant growth

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Exogenous addition of Spd and Spm increased plant biomass about 43% with the same

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effect in shoots and roots (Fig. 1). Under salt stress conditions, the effect of PAs

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treatment was similar on the SDW and higher on RDW which incremented by 57%. Salt

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treatment provoked a plant growth inhibition more significant in roots than in shoots

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with a 40% and 20% reduction, respectively. Pre-treatments with PAs did not modify

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the response to salinity in shoots and roots.

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3.2 Nitrogenase activity is increased by exogenous PAs

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Nitrogen fixation rate (NFR), evaluated through the nitrogenase activity and the nodule

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fresh weight (NFW) is shown in Fig. 2. In general, NFR was highly increased by the

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Spd treatment with 2.6 fold increment. Additionally, under salt stress conditions the

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effect of both PAs was even greater, which led to a reduction of the salinity inhibitory

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effect from 11 to 10 and 3 times with Spd and Spm, respectively. Nodule biomass was

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also induced by the Spd and Spm treatments with 44% and 32% increment,

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respectively. This effect was even higher under salt stress with double nodule biomass

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in plants co-treated with PAs and NaCl. Salinity provoked a NFW reduction of 40%

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while this effect was reduced to 20% with Spd and 4% with Spm.

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3.3 Nodule specific PAs accumulate under salt stress

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Content of PAs in leaves and nodules were analyzed in order to compare the effect of

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exogenous Spd and Spm in both tissues under salt stress as shown in Fig. 3. In general,

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the levels of PAs were higher in nodules, which also display a more complex PA profile

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because they contain nodule-specific PAs such as cadaverine (Cad) and

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homospermidine (Homspd). Exogenous Spd and Spm treatments provoked an

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increment of both PAs of about 50% in leaves. Under salt stress conditions, Spm was

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the only polyamine that accumulated in leaves, with 30% and 46% increment in control

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and Spm treated plants. Put levels did not show differences by PAs treatment in leaves

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but NaCl provoked a reduction that ranged from 50% to 2.7 fold. Spd showed a similar

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response to salt stress while Spm was the only polyamine that accumulated in salt-

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stressed leaves. A similar result was detected for Spm in nodules, although in addition

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to this polyamine, Cad doubled its concentration under salinity, independently of the co-

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treatment with exogenous Spd and Spm. Homspd was the most abundant polyamine in

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nodules and under salt stress increased between 37% and 16% depending on the

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exogenous polyamine treatment. Put and Spd responses to salinity in nodules were

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similar to the observed in leaves with significant reductions under salt stress conditions.

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3.4 Proline accumulation is partially prevented by exogenous Spm

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ACCEPTED MANUSCRIPT Proline (Pro) accumulation in leaves was quantified due to the close relation between

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the PAs metabolism and the synthesis of this amino acid. Salt stress induced a dramatic

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increase in the Pro levels in leaves with 3.6 fold higher concentration in such conditions

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(Fig. 4). However, the co-treatment with Spm partially inhibited the accumulation of

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Pro, which only showed a 76% induction by the salinity compared with the 3.6 fold

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induction observed in the absence of the PAs co-treatment. On the contrary, Spd did not

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induce any effect on the Pro accumulation by the salinity.

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3.5 Lipid peroxidation is inhibited by exogenous PAs

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Lipid peroxidation, estimated as the malondialdehyde (MDA) concentration in leaves

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(Fig. 5), was prevented by the exogenous treatment with Spd and Spm. Salt stress

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provoked a 2.5 fold increment in the MDA levels, however, with the exogenous Spd

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and Spm treatment this increment was 25% and 43%, respectively. In control

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conditions, no differences in the MDA levels were induced by the exogenous treatments

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with PAs.

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3.6 H2O2 production is reduced by exogenous PAs under salt stress

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H2O2 content in leaves was evaluated in a quantitative and in a semi-quantitative way in

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leaves as shown in Fig. 6A, B. The quantitative assay revealed that the level of H2O2

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was incremented by 3 times under salt stress conditions. However, pre-treatments with

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PAs reduced this increment to 30% with Spd and it was even completely abolished with

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Spm. Interestingly, the treatment with Spd and Spm produced an induction of H2O2 of

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40% and 2.5 times, respectively. The histochemical assay showed similar results to the

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quantitative assay with an increment of H2O2 in the salt stressed leaves and a slight

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reduction of this effect by the pretreatment with Spd.

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3.7 Exogenous PAs induce the synthesis of Brassinosteroids

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Expression level of genes involved in the synthesis of BRs DET2 (De-etiolated 2),

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DWF4 (Dwarf 4) and CYP85 were analyzed in leaves in order to determine a possible

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interaction between PAs and BRs metabolism (Fig. 7). Additionally, the expression of

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PAO1 (Polyamine oxidase 1) responsible of the Spd and Spm catabolism was analyzed.

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In general, all genes implicated in the synthesis of BRs were induced under salt stress

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conditions with 2 to 5 fold increment in the expression level. Exogenous treatments

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with PAs also induced the transcription level of the BRs biosynthetic genes to a higher

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extend with Spm which incremented 8 to 17 times the expression of the three genes

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analyzed. Nevertheless, the positive effect of Spd on the expression levels was

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completely abolished by salt stress and partially by the Spm treatment, except for

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DWF4 that did not show a significant difference. PAO1 transcription level was induced

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by Spd and Spm as well, while no significant differences were induced by the salt

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treatment.

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4. Discussion

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Exogenous application of PAs has been extensively shown to enhance tolerance to

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salinity stress in plants by scavenging free radicals, stabilizing membrane and cellular

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structures and modulating ion channels among other effects [25]; [3]; [26]. The positive

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effect of PAs has been associated to a cross-talk with other anti-stress hormones such as

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ACCEPTED MANUSCRIPT abcisic acid [27]; [28] salicylic acid [29], ethylene [30] or BRs [10, 12]. Indeed, the

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establishment of symbiosis between legumes and rhizobium has been shown to be

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influenced by the regulation of the PAs levels by a systemic effect of BRs on root

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nodule formation [15]. Those previous discoveries led us to study the effect of

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exogenous PAs (Spd and Spm) on the response to salinity of M. truncatula-S. meliloti

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symbiosis and its possible interaction with the BRs biosynthetic genes.

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Treatments with Spd and Spm increased plant growth, having similar effects on SDW

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and RDW (Fig. 1). This effect of exogenous PAs would be related with their growth

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regulatory capacity which includes DNA replication, cell division and root growth [3]

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Together with the increment in plant biomass, exogenous PAs incremented also nodule

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nitrogenase activity (Fig. 2).This could be also involved in the plant growth promotion

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due to a higher nitrogen supply to the plant. In that sense, a linear correlation between

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the concentration of PAs in nodules and nitrogenase activity has been shown [31].The

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increment of nitrogenase activity was accompanied by an increment of the nodule fresh

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weight as previously shown in Galega orientalis-Rhizobium galegae symbiosis [32].

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This effect could be related to a regulatory role of PAs in the nodulation process, as

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suggested by [33] in Lotus japonicus, where the expression of PAs biosynthetic genes

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(Spds and Spms) was maximal at early stages of nodule development.

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Under salt stress conditions Put and Spd levels were lower in leaves independently of

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the exogenous addition of PAs, while Spm augmented 32% in control plants and 46% in

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Spm treated plants (Fig. 3). In general, an increment in the (Spd+Spm)/Put ratio has

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been reported to be related with salinity tolerance in most cases [34]. In addition, Spm

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seems to participate on membrane modulation of permeability and stability [35]

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reducing radical generation under oxidative stress [36]. In that sense, Spm accumulation

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has been considered a general feature of plant response to salinity [37]. Exogenous Spd

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ACCEPTED MANUSCRIPT and Spm treatments increased foliar Spd and Spm levels by 50% and 70%, respectively,

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which indicates a transport of these PAs from the root to the shoot. Nodule PAs analysis

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revealed the presence of uncommon PAs such as Cad and Homspd, characteristics of

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nodules of legumes such as M. truncatula [38] or Phaseolus vulgaris [9]. In addition,

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the levels of all PAs were higher in nodules than in leaves, as described by [39], with

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Homspd as the most abundant PA synthesized by the bacteroids [38]. Similarly to

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leaves, exogenous Spd and Spm treatments incremented the nodular content of both

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PAs which correlated with an increment of the nodule biomass (NFW) and nitrogenase

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activity (NFR), suggesting the implication of Spd and Spm in the improvement of the

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symbiosis effectiveness. Nevertheless, Cad and Homspd levels declined by the

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exogenous addition of the common PAs (Spd and Spm), which might be due to the

337

replacement of these uncommon PAs by Spd and Spm, since the exact structure of these

338

compounds has been shown not to be critical for their function [40]. Interestingly, the

339

concentration of nodule specific PAs (Cad and Homspd) augmented under salt stress

340

due to the implication of Homspd in the stress tolerance of fast-growing rhizobia [41].

341

Proline accumulation is considered one of the most commonly adaptive responses of

342

plants to salinity due to its antioxidant and osmo-compatible properties [42].

343

Additionally, polyamine metabolism exerts a direct or indirect action on proline

344

metabolism through PAs degradation, which could contribute to proline accumulation,

345

or by the competition of both metabolites by glutamate as common precursor [43]. In

346

this context, salt stress strongly induced proline accumulation in M. truncatula leaves

347

(Fig 4) which suggest prevalence of this amino acid over PAs in the response to salinity

348

as previously described in Medicago sativa nodules [43]. Nevertheless, exogenous Spm

349

treatment partially prevented proline accumulation which together to the Spm

350

accumulation under salinity suggests that in such conditions, this polyamine could

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16

ACCEPTED MANUSCRIPT replace the role of proline without requiring its accumulation. This result is supported

352

by [44] who reported that exogenous PAs decreased Pro accumulation in salinity

353

exposed Phaseolus vulgaris plants.

354

In addition to the primary effects, salinity leads to oxidative stress through an increase

355

of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), which cause lipid

356

peroxidation and disturb cellular membrane functioning. The H2O2 level increased

357

significantly upon salt stress exposure (Fig. 6A), which indicated higher oxidative stress

358

in these plants. MDA, a lipid peroxidation product, served as the indicator of the extent

359

oxidative damage in salt stressed plants in this study (Fig. 5). Spd and Spm pre-

360

treatments reduced the levels of H2O2 and MDA during salt stress. These results agree

361

with previous studies [25]; [45] and suggest that PAs serve as efficient ROS scavenger

362

and membrane stabilizers by the MDA reduction under salt stress. However, at low

363

concentrations, H2O2 is known to prevent oxidative damage by up-regulating

364

antioxidative defence enzymes and by restoring redox homeostasis [46] which make

365

vital to keep the level of H2O2 rather than removing it. In that sense, visual

366

identification of H2O2 detected by histochemical staining (Fig. 6B) as well as the

367

quantitative analysis reflected a moderate increment of H2O2 in Spd and Spm treated

368

plants.

369

The beneficial effects of PAs in the response to salinity have been associated to a cross-

370

talk with other plant growth regulators such as BRs [47]. Indeed, in a previous study we

371

found that the salt stress ameliorative effect of 24-epibrassinolide in the M. truncatula-

372

S. meliloti symbiosis was mediated by PAs [10]. In that sense, we found that exogenous

373

PAs induced the expression of genes involved in the BRs biosynthesis (Fig. 7) which

374

support a cross-talk between PAs and BRs in the salt stress response of the M.

375

truncatula-S. meliloti symbiosis.

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ACCEPTED MANUSCRIPT In conclusion, exogenous PAs improved the response to salinity of the M. truncatula-S.

377

meliloti symbiosis by reducing the nitrogenase inhibition as well as the oxidative

378

damage induced under salt stress conditions. Additionally, in this work we provide

379

evidences of the cross-talk between PAs and BRs in the adaptive responses to salinity.

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380

Aknowledgements

382

This work has been supported by the Andalusian Research Program (AGR-139) and the

383

Spanish Ministry of Science and Technology (Grant: AGL2013-42778-P).

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ACCEPTED MANUSCRIPT Figure legends

397

Figure 1

398

Effect of exogenous Spd and Spm on shoot dry weight (SDW) and root dry weight

399

(RDW) of M. truncatula plants inoculated with S. meliloti under control (white bars)

400

and salt stress conditions (grey bars). Data are means + SE (n=10). Mean values

401

followed by the same letter do not differ (p<0.05) using the LSD test.

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Figure 2

404

Effect of exogenous Spd and Spm on nitrogen fixation rate (NFR) and nodule fresh

405

weight (NFW) of M. truncatula plants inoculated with S. meliloti under control (white

406

bars) and salt stress conditions (grey bars). Data are means + SE (n=10). Mean values

407

followed by the same letter do not differ (p<0.05) using the LSD test.

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Figure 3

410

Effect of exogenous Spd and Spm on polyamines content (Put, putrescine; Spd,

411

spermidine; Spm, spermine; Cad, cadaverine; Homspd, homospermidine) in M.

412

truncatula leaves (L) and nodules (N) inoculated with S. meliloti under control (white

413

bars) and salt stress conditions (grey bars). Data are means + SE (n=4). Mean values

414

followed by the same letter do not differ (p<0.05) using the LSD test.

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Figure 4

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ACCEPTED MANUSCRIPT Effect of exogenous Spd and Spm on proline content (Pro) in leaves of M. truncatula

418

inoculated with S. meliloti under control (white bars) and salt stress conditions (grey

419

bars). Data are means + SE (n=4). Mean values followed by the same letter do not differ

420

(p<0.05) using the LSD test.

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Figure 5

423

Effect of exogenous Spd and Spm on malondialdehyde content (MDA) in leaves of M.

424

truncatula inoculated with S. meliloti under control (white bars) and salt stress

425

conditions (grey bars). Data are means + SE (n=4). Mean values followed by the same

426

letter do not differ (p<0.05) using the LSD test.

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427

Figure 6

429

Effect of exogenous Spd and Spm on (A) H2O2 content in leaves of M. truncatula

430

inoculated with S. meliloti under control (white bars) and salt stress conditions (grey

431

bars). Data are means + SE (n=4). Mean values followed by the same letter do not differ

432

(p<0.05) using the LSD test. (B) DAB staining of H2O2 in leaves of M. truncatula

433

inoculated with S. meliloti and treated or not with NaCl and Spd.

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428

435

Figure 7

436

Effect of exogenous Spd and Spm on expression level of DET2, DWF4, CYP85 and

437

PAO1 genes in leaves of M. truncatula inoculated with S. meliloti under control (white

20

ACCEPTED MANUSCRIPT 438

bars) and salt stress conditions (grey bars). Data are means + SE (n=4). Mean values

439

followed by the same letter do not differ (p<0.05) using the LSD test.

440

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ACCEPTED MANUSCRIPT References

443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491

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[39] S. Fujihara, H. Abe, Y. Minakawa, S. Akao, T. Yoneyama, Polyamines in nodules from various plant-microbe symbiotic associations, Plant and Cell Physiology, 35 (1994) 11271134. [40] F.L. Shaw, K.A. Elliott, L.N. Kinch, C. Fuell, M.A. Phillips, A.J. Michael, Evolution and Multifarious Horizontal Transfer of an Alternative Biosynthetic Pathway for the Alternative Polyamine sym-Homospermidine, J. Biol. Chem., 285 (2010) 14711-14723. [41] S. Fujihara, Biogenic Amines in Rhizobia and Legume Root Nodules, Microbes and Environments, 24 (2009) 1-13. [42] A. Aziz, J. Martin-Tanguy, F. Larher, Salt stress-induced proline accumulation and changes in tyramine and polyamine levels are linked to ionic adjustment in tomato leaf discs, Plant Science, 145 (1999) 83-91. [43] M. Lopez-Gomez, J. Hidalgo-Castellanos, C. Iribarne, C. Lluch, Proline accumulation has prevalence over polyamines in nodules of Medicago sativa in symbiosis with Sinorhizobium meliloti during the initial response to salinity, Plant Soil, 374 (2014) 149-159. [44] J.F. Jimenez-Bremont, A. Becerra-Flora, E. Hernandez-Lucero, M. Rodriguez-Kessler, J.A. Acosta-Gallegos, J.G. Ramirez-Pimentel, Proline accumulation in two bean cultivars under salt stress and the effect of polyamines and ornithine, Biol. Plantarum, 50 (2006) 763-766. [45] K. Nahar, M. Hasanuzzaman, A. Rahman, M. Alam, J.A. Mahmud, T. Suzuki, M. Fujita, Polyamines confer salt tolerance in mung bean (Vigna Radiata l.) by reducing sodium uptake, improving nutrient homeostasis, antioxidant defense, and Methylglyoxal detoxification systems, Frontiers in Plant Science, 7 (2016). [46] S. Bhattacharjee, An inductive pulse of hydrogen peroxide pretreatment restores redoxhomeostasis and oxidative membrane damage under extremes of temperature in two rice cultivars, Plant Growth Regulation, 68 (2012) 395-410. [47] Q. Fariduddin, B.A. Mir, M. Yusuf, A. Ahmad, Comparative roles of brassinosteroids and polyamines in salt stress tolerance, Acta Physiol. Plant., 35 (2013) 2037-2053.

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ACCEPTED MANUSCRIPT 578 Table 1. Primer sequences of Medicago truncatula genes used for qRT-PCR. Oligonucleotide sequence

Product size

RI PT

Gene

Fw 5´-GTCGCGGTGGTTTGAATTCC-3´ Mt DET2

154 bp

SC

Rv 5´-AGTCCCAAATTCGGCATACC-3´

Fw 5´-TTCCATGTGGGTGGAAAGTC-3´ Mt DWF4

171 bp

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Rv 5´-GATCCTGCGCATAATCTTGG-3´

Fw 5´-CTGTGAAGTACCTCCATGAC-3´ Mt CYP85

162 bp

TE D

Rv 5´-GTGGCCAATCTAGTTGTCTC-3´

Fw 5´-GGCCTGACGTAGAACTTATG-3´

Mt PAO1

312 bp

EP

Rv 5´-GCACGTAGCTTGTCATACAC-3´

Fw 5´-ATGGGGCAGAAGGATGCGTATG-3´ 115 bp

AC C

Mt Actin

Rv 5´-AGCCTTCATAGATGGGGACCGT-3´

579

580

581

582

25

ACCEPTED MANUSCRIPT 583

Figure 1

584 0,30

SDW PSPA aa

587

aa

0,20

g plant-1

586

0,15

ab

bb

ab

c

b 0,06

0,10 0,05 0,00

0,00 Spd

Spm

C

M AN U

590 591

598

EP AC C

597

TE D

592

596

b

SC

C

595

a b

c

0,03

589

594

a

0,09

588

593

RDW PSR

0,12

RI PT

0,25

g plant-1

585

0,15

599 600 601

26

Spd

Spm

ACCEPTED MANUSCRIPT 602

Figure 2

603

NFR

b b

607 c

c

610

616 617

EP

615

AC C

614

25

c

C

Spm

TE D

611

613

b

b

M AN U

Spd

609

612

b b

0 C

a

c

0

608

50

RI PT

a

100

50

NFW mg plant-1

606

µmol N g-1 NFW h-1

605

75

150

SC

604

618 619 620

27

Spd

Spm

ACCEPTED MANUSCRIPT 621

Figure 3

622 623

5

AB

1,0

B

a a

a C

ab

0,5

b

C

C

b

4

a

0,0

b

2

SPM

Spd Spm

D

F E

C

638

C

Spd Spm

N

B C D E F

0,0 C

Spd Spm

L

nmols g-1 FW

AC C

HOMSPD A B C

6

D

D E

4 2 0 Spd Spm

L

C

Spd Spm

N

28

C

Spd Spm

N

10 8

A

CAD

0,5

N

C

639

Spd Spm

1,0

Spd Spm

EP

C

C

TE D

cd d

nmols g-1 FW

b c

L

637

D

b

L

B

a

1,0

0,0

636

E

A

1,5

632

635

C

N

0,5

634

C

1,5

d

633

Spd Spm

2,0

nmols g-1 FW

631

C

L

629 630

Spd Spm

b

M AN U

C

B

b

b

0

628

B

3

1

627

A

SPD

RI PT

626

PUT

SC

625

6 A

nmols g-1 FW

624

nmols g-1 FW

1,5

ACCEPTED MANUSCRIPT 640

Figure 4

641 642

PRO

643

645

a

a 1000

b 500

c

c

646 C

647 648

652 653 654 655

EP

651

AC C

650

Spd

TE D

649

c

M AN U

0

SC

644

nmol gFW -1

1500

RI PT

2000

656 657 658

29

Spm

ACCEPTED MANUSCRIPT 659

Figure 5

660 661

800

b 400

c

c

200

664

0

665 666 667

673 674

EP

672

AC C

671

TE D

668

670

Spd

c

Spm

M AN U

C

669

c

SC

µmol MDA gFW-1

663

600

RI PT

MDA

a 662

675 676 677

30

ACCEPTED MANUSCRIPT 678

Figure 6

679

A 680

20

µmol gFW -1

15

682

a b

10

5

684

0

d

685

B

TE D

687

689

Spd

692 693

AC C

691

NaCl

EP

Control 690

Spm

M AN U

C

688

bc

c

683

686

b

SC

681

RI PT

H2O2

Spd + NaCl

Spd

694 695 696

31

ACCEPTED MANUSCRIPT 697

Figure 7

698

25

25

DET2 20

10

c

5

Spd

CYP85

20 15

20

a

10

b

5

c

TE D

Fol change

C

a

b

c

Spd

Spm

25

707

b

b

0

Spd

b

Spm

AC C

EP

C

712

Spm

Fol change

705

b c

SC

C

25

711

10

0

704

710

a

5

0

703

709

15

d

d

708

Fol change

b

15

DWF4

20

c

702

706

a

M AN U

701

Fol change

700

RI PT

699

713

32

PAO1

15 10

a a

a

5

b b

b

0 C

Spd

Spm

ACCEPTED MANUSCRIPT Highlights 1. Exogenous polyamines increment nodule biomass and nitrogenase activity in the symbiosis Medicago truncatula-Sinorhizobium meliloti.

peroxidation under salt stress.

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2. Spermidine and spermine pre-treatments reduce the levels of H2O2 and lipid

3. Polyamines induce the expression of genes involved in brassinosteroids biosynthesis.

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4. Exogenous polyamines improve the response to salinity of the M.

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truncatula-S. meliloti symbiosis by reducing the oxidative damage.

ACCEPTED MANUSCRIPT Contribution

Miguel López Gómez: conceived and designed the experiments and wrote the

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manuscript. Javier Hidalgo-Castellanos: performed the experiments and analyzed the data.

SC

J. Rubén Muñoz-Sánchez: performed some of the experiments

Carmen Lluch Plá: Project leader

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Agustín J. Marín-Peña: performed some of the experiments

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José A. Herrera-Cervera: Project leader