Cyclophilin AtROC1^S58F confers Arabidopsis cold tolerance by modulating jasmonic acid signaling and antioxidant metabolism

Highlights

• Gene Ontology terms ‘response to jasmonic acid stimulus’ and ‘response to oxidative stress’ were identified significantly enriched in Cyclophilin ROC1^S58F mutant.

• ROC1^S58F showed freezing cold tolerance associated with increased survival rate and a reduced level of electrolyte leakage and endogenous JA content.

• Down-regulated of JA biosynthesis and signaling genes, and increased ROS-scavenging enzyme activities and gene expression were identified in cold-tolerance ROC1^S58F mutant.

Abstract

Cyclophilins (CYPs), a class of proteins with a conserved peptidyl-prolyl cis-trans isomerase domain, are widely involved in the regulation of plant growth and development, as well as in the response to abiotic stresses including cold. In our previous study, we identified an Arabidopsis gain-of-function mutant ROC1^S58F with enhanced cold-tolerance and enhanced expression of jasmonic acid (JA) and oxidative stress responsive genes. Here, we show the underlying molecular mechanisms for the improved cold tolerance observed in the ROC1^S58F mutant. Compared to the WT, the ROC1^S58F mutant showed an increased survival rates and a reduced level of electrolyte leakage and endogenous JA content under the freezing treatment. Correspondingly, the JA biosynthesis genes (AtAOC1 and AtOPR3) and signaling genes (AtJAZ5, AtJAZ10 and AtMYB15) are down-regulated in the ROC1^S58F mutant compared with the WT. Moreover, both the transcripts and activities of the ROS-scavenging enzymes (SOD/POD/MDHAR) increased in cold-stressed ROC1^S58F mutant, which might mitigate the ROS-induced oxidative stress and contribute to the mutant freezing tolerance. Taken together, our findings indicate that AtROC1^S58F confers Arabidopsis freezing tolerance by modulating JA signaling and antioxidant metabolism jointly. This research thus provides a molecular mechanism for AtROC1^S58F-conferred freezing resistance in Arabidopsis and offers guidance for crop breeding towards an improved cold tolerance.

Introduction

Cold stress is one of the most devastating abiotic stresses that affect plant growth, productivity, and distribution. Extremely low temperature could disrupt cellular homeostasis and induce the accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), superoxide anion (O₂⁻), and hydroxyl radical (Sinha et al., 2015). Correspondingly, plants have evolved sophisticated mechanisms to mitigate cellular oxidative damages caused by cold stress, including physiological and biochemical modifications and the activation of cold-responsive genes and transcription factors (TFs). Specifically, antioxidant enzymes (e.g., superoxide dismutase [SOD], peroxidase [POD], monodehydroascorbate reductase [MDHAR], glutathione reductase [GR]) and non-enzymatic antioxidants including ascorbic acid (ASA) and glutathione (GSH) accumulate to detoxify ROS accumulation (Mittler et al., 2004).

Cyclophilins (CYPs), a class of proteins that contain the conserved peptidyl-prolyl isomerase (PPIase) domain, are involved in protein folding and are known to play essential roles in various processes of plant growth and development (Dipesh Kumar et al., 2012). For example, AtCYP40 interacts with Hsp 90 to regulate the number of juvenile leaves and the morphology of inflorescence in Arabidopsis (Berardini et al., 2001). The Arabidopsis CYP71 (AtCYP71) and rice CYP2 (OsCYP2) both participate in lateral development, while the former being a histone remodeling factor that affects meristem cell organization in Arabidopsis (Li and Luan, 2011) and the latter regulating the stability of OsIAA11 (Indole-3-acetic acid) to control lateral root development in rice (Oryza sativa) (Jing et al., 2015; Kang et al., 2013). In addition, overexpression of AtCYP20-2 and TaCYP20-2 alters Arabidopsis flowering time (Zhang et al., 2013). The chloroplast stroma-localized protein AtCYP20-3 interacts with serine acetyltransferase to respond to high light and oxidative stress in Arabidopsis (Dominguez-Solis et al., 2008) and overexpression of OsCYP20-2 confers tobacco (Nicotiana tabacum) resistance to osmotic stress and high light (Kim et al., 2012). Furthermore, OsCYP19-4 exerts cold-tolerance and is associated with increased tiller and spike numbers in rice (Yoon et al., 2016). Despite their importance in cold tolerance and growth regulation, the detailed mechanisms employed by CYPs in response to cold stress are still unclear.

Jasmonic acids (JAs) are a class of plant phytohormones essential for plant development and stress tolerance (Sharma and Laxmi, 2016). Previous studies have shown that CYPs likely confer plant cold tolerance through JA signaling, which is known to participate in the response to a broad spectrum of stress conditions (Chen et al., 2007; Trivedi et al., 2013). In plants, JAs are synthesized from theα-linolenlic acid (18:3) in the chloroplasts membranes, which was then catalyzed mainly by lipoxygenases, allene oxide synthase (AOS), allene oxide cyclase (AOC), and 12-oxophytodienoate reductase 3 (OPR3), in plastids and peroxisomes (Santino et al., 2013). JA signaling is initiated by the formation of a co-receptor complex consisting of JA receptor coronatine insensitive 1 (COI1) and jasmonate-ZIM domain (JAZ) repressor, where JAZ is ubiquitinated and degraded by the 26S proteasome, thereby allowing for the binding of MYC transcriptional factors to the G-box element that functions downstream of JA-responsive genes (Per et al., 2018). In cold-stressed rice plants, endogenous JA level and the transcript levels of JA biosynthetic genes OsIOX2, OsAOC, and OsAOS1 were significantly increased (Du et al., 2013); and exogenous methyl jasmonate application significantly enhances the freezing tolerance of Arabidopsis plants (Hu et al., 2013). Further research indicated that exogenous applied JA could increase ASA content and POD enzymatic activity in carambola (Averrhoa carambola) during cold storage (Mustafa et al., 2016). These results suggest that jasmonate might play important roles in imparting cold tolerance in plants.

AtCYP18-3 (ROC1, Rotamase CYP 1) is a member of the CYP family. In our previous study, we identified a gain-of-function Arabidopsis roc1 mutant ROC1^S58F, which exhibited substantially increased sensitivity to temperature (Ma et al., 2013). The ROC1^S58F mutant does not bolt and sets only a few flowers from the center of the rosette under normal growth conditions, but produces an increasing number of flowers and normal siliques at a low temperature (16 °C) (Ma et al., 2013). Here, we investigated the underlying molecular mechanisms for these phenotypic changes under different temperatures, and hypothesized that abiotic stress level in the mutant was alleviated, likely associated with increases in endogenous hormone levels and antioxidant activities.