Research article 24-Epibrassinolide ameliorates salt stress effects in the symbiosis Medicago truncatula-Sinorhizobium meliloti and regulates the nodulation in cross-talk with polyamines Miguel L  opez-G  omez * , Javier Hidalgo-Castellanos, Carmen Lluch, Jos  e A. Herrera-Cervera Departamento de Fisiología Vegetal, Facultad de Ciencias, Universidad de Granada, Campus de Fuentenueva s/n,18071, Granada, Spain article info Article history: Received 21 June 2016 Received in revised form 14 July 2016 Accepted 18 July 2016 Available online 19 July 2016 Keywords: Brassinosteroids 24-Epibrassinolide Polyamines Salt stress Symbiosis Medicago truncatula abstract Brassinosteroids (BRs) are steroid plant hormones that have been shown to be involved in the response to salt stress in cross-talk with other plant growth regulators such as polyamines (PAs). In addition, BRs are involved in the regulation of the nodulation in the rhizobium-legume symbiosis through the alter- ation of the PAs content in leaves. In this work, we have studied the effect of exogenous 24- epibrassinolide (EBL) in the response to salinity of nitrogen fixation in the symbiosis Medicago truncatula-Sinorhizobium meliloti. Foliar spraying of EBL restored the growth of plants subjected to salt stress and provoked an increment of the nitrogenase activity. In general, PAs levels in leaves and nodules decreased by the salt and EBL treatments, however, the co-treatment with NaCl and EBL augmented the foliar spermine (Spm) concentration. This increment of the Spm levels was followed by a reduction of the membrane oxidative damage and a diminution of the proline accumulation. The effect of BRs on the symbiotic interaction was evaluated by the addition of 0.01, 0.1 and 0.5 mM EBL to the growing solution, which provoked a reduction of the nodule number and an increment of the PAs levels in shoot. In conclusion, foliar treatment with EBL had a protective effect against salt stress in the M. truncatula-S. meliloti symbiosis mediated by an increment of the Spm levels. Treatment of roots with EBL incre- mented PAs levels in shoot and reduced the nodule number which suggests a cross-talk between PAs and BRs in the nodule suppression and the protection against salt stress. © 2016 Elsevier Masson SAS. All rights reserved. 1. Introduction Brassinosteroids (BRs) are polyhydroxilated steroid hormones that regulate various aspects of plant growth and development including cell elongation, tissue differentiation (Gudesblat and Russinova, 2011) and abiotic stress responses including salt stress (Bajguz, 2011; Ali et al., 2007). In previous studies the involvement of BRs in plant stress resistance by the improvement of antioxidant capacity (El-Mashad and Mohamed, 2012; Sharma et al., 2015), ion homeostasis (El-Khallal et al., 2009) and promotion of osmolytes accumulation (Ragab and Ali, 2006) has been determined. BRs biosynthesis occurs through a network of pathways and basically is regulated at the transcriptional level of the biosynthetic genes, although the regulatory mechanisms responsible for the BRs homeostasis remain largely unknown. In that sense, BRs biosynthesis has been shown to be affected by other hormones such as auxin (Chung et al., 2011) and a crosstalk between BRs and other plant growth regulator such as polyamines (PAs) has also been described in the response to abiotic stress (Zheng et al., 2016; Choudhary et al., 2012a; Choudhary et al., 2011). PAs such as putrescine (Put), spermidine (Spd) and sper- mine (Spm) are small aliphatic nitrogen compounds found in all living cells including plant cells, and have been found to be involved in a wide range of biological processes such as cell divi- sion and differentiation (Hussain et al., 2011) or the protection against a variety of environmental stresses including salinity (L opez-G omez et al., 2014). Legumes are considered salt sensitive crop species due to the Abbreviations: EBL, 24-epibrassinolide; BRs, brassinosteroids; PAs, polyamines; Put, putrescine; Spd, spermidine; Spm, spermine; Cad, cadaverine; Homspd, homospermidine; MDA, malondialdehyde; Pro, proline; ADC, arginine decarbox- ilase; SPDS, spermidine synthase; SPMS, spermine synthase; NFR, nitrogen fixation rate; EAC, electron allocation coefficient; NFW, nodule fresh weight; ANA, apparent nitrogenase activity; TNA, total nitrogenase activity. * Corresponding author. E-mail address: mlgomez@ugr.es (M. L opez-G omez). Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy http://dx.doi.org/10.1016/j.plaphy.2016.07.017 0981-9428/© 2016 Elsevier Masson SAS. All rights reserved. Plant Physiology and Biochemistry 108 (2016) 212e221