Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Overexpression of 14-3-3 proteins enhances cold tolerance and increases levels of stress-responsive proteins of Arabidopsis plants Sabina Visconti a, ⁎ , Chiara D’Ambrosio b, ⁎⁎ , Anna Fiorillo a , Simona Arena b , Carlo Muzi a , Michela Zottini c , Patrizia Aducci a , Mauro Marra a , Andrea Scaloni b , Lorenzo Camoni a a Department of Biology, University of Rome Tor Vergata, 00133, Rome, Italy b Proteomics & Mass Spectrometry Laboratory ISPAAM, National Research Council, 80147, Naples, Italy c Department of Biology, University of Padova, 35131, Padova, Italy ARTICLE INFO Keywords: 14-3-3 Proteins Plant proteomics H + -ATPase Cold stress ABSTRACT 14-3-3 proteins are a family of conserved proteins present in eukaryotes as several isoforms, playing a regulatory role in many cellular and physiological processes. In plants, 14-3-3 proteins have been reported to be involved in the response to stress conditions, such as drought, salt and cold. In the present study, 14-3-3ε and 14-3-3ω isoforms, which were representative of ε and non-ε phylogenetic groups, were overexpressed in Arabidopsis thaliana plants; the effect of their overexpression was investigated on H + -ATPase activation and plant response to cold stress. Results demonstrated that H + -ATPase activity was increased in 14-3-3ω-overexpressing plants, whereas overexpression of both 14-3-3 isoforms brought about cold stress tolerance, which was evaluated through ion leakage, lipid peroxidation, osmolyte synthesis, and ROS production assays. A dedicated tandem mass tag (TMT)-based proteomic analysis demonstrated that different proteins involved in the plant response to cold or oxidative stress were over-represented in 14-3-3ε-overexpressing plants. 1. Introduction 14-3-3 proteins are a family of conserved proteins in eukaryotes that play a regulatory role in many cellular and physiological processes by direct interaction with target proteins [1]. They bind specific phos- phoserine/phosphothreonine containing motifs [2–4] in the clients, thus affecting their activity, subcellular localization or stability [5,6]. In plants, 14-3-3 proteins are involved in the regulation of ion membrane transport, carbon and nitrogen metabolism, gene expression, stomatal movement, hormone signaling and in the coordination of different signal transduction pathways [7–11]. They have also been reported to be involved in the plant response to stress conditions [12–14]. In fact, environmental and biotic stimuli affect the expression levels of 14-3-3 proteins. Furthermore, many proteins involved in the response to dif- ferent stresses have been shown to be 14-3-3 clients [14–16]. In par- ticular, 14-3-3 proteins play a role in the plant response to water stress, as demonstrated by the improvement of tolerance under moderate drought stress conditions displayed by cotton plants overexpressing the Arabidopsis 14-3-3λ [17]. Similarly, the maize ZmGF14-6 isoform, constitutively expressed in rice, enhanced tolerance to drought by strongly inducing drought-responsive rice genes [18]. Recently, 14-3-3 proteins have been reported to be involved in the salt overly sensitive (SOS) pathway, which is known to control sodium homeostasis during salt stress [19]. Accordingly, the expression of 14-3-3 isoforms BdGF14d and TaGF14b from Brachypodium dystachion and wheat, re- spectively, was shown to enhance salt tolerance in transgenic tobacco plants [20,21]. It has also been demonstrated that 14-3-3 proteins take part to the response to cold stress; in fact, Jarillo and colleagues identified in Arabidopsis two 14-3-3 proteins, namely RARE COLD INDUCIBLE 1A and 1B, which are induced by cold stress [22]. More recently, the Arabidopsis 14-3-3ψ isoform, i.e. RCI1A, has been demonstrated to function as a negative regulator of cold acclimation and freezing tol- erance, by negatively controlling the expression of cold-responsive genes [23]. Similarly, Liu and coworkers demonstrated the involvement of the Arabidopsis 14-3-3λ isoform in the negative regulation of the stability of C-repeat binding factor (CBF) proteins that play a pivotal role in freezing tolerance [24]. 14-3-3 proteins exist as multiple isoforms; eight isoforms are ex- pressed in rice [25], twelve in tomato [26], and thirteen in Arabidopsis [27], where they are designed by Greek letters and divided into two major groups (named ε and non-ε), based on phylogenetic analysis. The https://doi.org/10.1016/j.plantsci.2019.110215 Received 18 April 2019; Received in revised form 22 July 2019; Accepted 6 August 2019 ⁎ Corresponding author at: Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133, Rome, Italy. ⁎⁎ Corresponding author at: Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, Via Argine 1085, 80147, Napoli, Italy. E-mail addresses: visconti@uniroma2.it (S. Visconti), chiara.dambrosio@cnr.it (C. D’Ambrosio). Plant Science 289 (2019) 110215 Available online 28 September 2019 0168-9452/ © 2019 Elsevier B.V. All rights reserved. T