A Universal Stress Protein from Medicago falcata (MfUSP1) confers multiple stress tolerance by regulating antioxidant defense and proline accumulation

Highlights

• MfUSP1 transcript is induced by ABA, H₂O₂ and multiple abiotic stresses.

• Overexpression of MfUSP1 leads to increased tolerance to multiple abiotic stresses.

• MfUSP1 expression upregulates antioxidant defense and proline accumulation.

Abstract

Universal Stress Proteins (USPs) are ubiquitously responsive to environmental stresses, but function of most of them remains largely unknown. A novel USP from Medicago falcata (MfUSP1) was identified in the present study. MfUSP1 transcript is induced by low temperature, salinity, osmotic stress, abscisic acid (ABA) and H₂O₂. Overexpression of MfUSP1 resulted in increased tolerance to multiple abiotic stresses including freezing, salinity, osmotic stress, and methyl viologen (MV) induced oxidative stress. Higher activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate dependent peroxidase (APX) were observed in transgenic plants than in the wild type (WT) under the stressed conditions with lower reactive oxygen species (ROS) accumulation. Compared to WT, higher transcript levels of the genes encoding Cu, Zn-SOD, CAT, and APX were observed in transgenic plants under salinity and osmotic stress, and higher transcript levels of CAT and APX encoding genes under low temperature condition. Moreover, more proline was accumulated in transgenic plants than in WT under the stressed conditions, with upregulation of pyrroline-5-carboxylate synthetase 2 (P5CS2) and downregulation of proline oxidase 1 and 2 (PROX1 and PROX2). It is suggested that MfUSP1 confers tolerance to multiple abiotic stresses by regulating antioxidant defense and proline accumulation under stressed conditions.

Introduction

Plants inevitably suffer from various abiotic stresses, such as drought, extreme temperature, salinity, heavy metals, and UV radiation (Bhatnagar-Mathur et al., 2008; Vishwakarma et al., 2017). Various environmental factors impose common challenges to plant cells with low water potential, excessive reactive oxygen species (ROS), and redox imbalance, which lead to protein denaturation, lipid peroxidation and organelle malfunction (Mehla et al., 2017; Kazemi-Shahandashti and Maali-Amiri, 2018; Yang and Guo, 2018; Sharma et al., 2019).

Osmotic adjustment and antioxidant defense system are important physiological mechanism in plant adaptation to environmental stresses. Plants accumulate a large amount of compatible osmolytes, such as proline, glycine betaine, mannitol, and soluble sugars, to maintain cell turgor and water availability under abiotic stresses (Chen and Jiang, 2010). Proline is an excellent multifunctional molecule, not only an osmolyte but also as ROS scavenger and stabilizer of proteins and membranes (Hayat et al., 2012). Pyrroline-5-carboxylate synthetase (P5CS) is the key enzyme for proline biosynthesis, while proline oxidase (PROX) catalyzes proline degradation in higher plants. Both biosynthesis and catabolism of proline are regulated by stresses (Szepesi and Szőllősi, 2018). Proline biosynthesis is usually enhanced under stresses via transient or continuous stimulation of P5CS expression, depending on severity and duration of the stress situation (Dobra et al., 2011; Nair et al., 2012; Iqbal et al., 2015; Feng et al., 2016). On the contrary, proline degradation is often downregulated as a result of reduced PROX expression or enzyme activity (Satoh et al., 2002; Sharma and Verslues, 2010; Ren et al., 2018). Antioxidant defense system is consisted of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR), and non-enzymatic antioxidants, such as ascorbic acid and reduced glutathione (GSH) (Sharma et al., 2019). SOD converts superoxide radical (O₂⁻) into H₂O₂, which can be further scavenged by CAT or APX. Ascorbic acid functions as a substrate of APX and is oxidized to dehydroascorbate (DHA) or monodehydroascrobate (MDHA) when H₂O₂ is scavenged, while DHA and MDGA can be reduced to ascorbic acid throught ascorbate-glutathione cycle being involved in GSH, GR, DHAR and MDHAR (Mehla et al., 2017). Antioxidants play a key role in scavenging the accumulated ROS when plants are exposed to abiotic stresses, which is important for plant survival under stress conditions.

The Universal Stress Protein (USP) was first found in Escherichia coli, in which a 13.5 kDa cytosolic protein was induced by a broad range of stresses (VanBogelen et al., 1990; Nystrom and Neidhardt, 1992). Increasing evidence indicated that USP homologs exist in bacteria, archaea, fungi, plants, and even in some invertebrates. The USP superfamily is prosperous in plants. There are usually 20 –50 members in a single plant genome, and even 142 in Brassica napus (Li et al., 2010; Chi et al., 2019). They are essential for colonization, pathogenicity and adaptation to oxidative stress, high temperature, low pH and hypoxia (Vollmer and Bark, 2018). Among 44 USP domain(s)-containing proteins in Arabidopsis, two USPs are involved in adaption to temperature stresses and anoxia (Kerk et al., 2003; Gonzali et al., 2015; Jung et al., 2015; Melencion et al., 2017). Two USPs mediate the alleviation of oxidative stress respectively in Solanum pennelli and Solanum lycopersium (Loukehaich et al., 2012; Gutierrez-Beltran et al., 2017). Overexpressing SbUSP from Salicornia brachiata promotes plant growth in transgenic tobacco under multiple stresses (Udawat et al., 2016). A number of stress-response USPs are found in diverse plants, such as rice, cotton, barley, Salvia miltiorrhiza, Gossypium arboreum and Astragalus sinicus (Sauter et al., 2002; Maqbool et al., 2009; Chou et al., 2007; Li et al., 2010; Wang et al., 2017). However, only a few plant USP homologs have been investigated.

Alfalfa (Medicago sativa ssp. sativa) is the most important forage legume with high yield and nutrient quality, while Medicago sativa ssp. falcata (hereafter M. falcata) is closely related to alfalfa with excellent cold and drought tolerance. It is important to understand the mechanisms of cold and drought tolerance mechanism in M. falcata and to identify the relevant genes for potential use in crop improvements. Several genes such as myo-inositol phosphate synthase (MfMIPS), galactinol synthase (MfGolS1), hybrid proline-rich protein (MfHyPRP), myo-inositol transporter-like (MfINT-like), and ethylene responsive factor (MfERF) in M. falcata have been identified to confer cold, drought and salt tolerance (Guo et al., 2014; He et al., 2015; Sambe et al., 2015; Zhuo et al., 2016, 2018). In the present study, a novel MfUSP1 gene was characterized, and its role in multiple stress tolerance was investigated using transgenic tobacco plants. Our data suggest that MfUSP1 confers multiple stress tolerance by upregulating proline accumulation and antioxidant defense under stressed conditions.