SELENOPROTEIN O is a chloroplast protein involved in ROS scavenging and its absence increases dehydration tolerance in Arabidopsis thaliana
Introduction
Plants respond to environmental changes by altering many molecular and physiological processes. These responses are tightly controlled at cellular, organ and whole plant levels. Key components in this interplay are redox regulation and generation of reactive oxygen species (ROS) [1]. Changes in redox homeostasis affect protein structure and enzymatic activity, and influence signal transduction [2]. In plants, most of ROS production is modulated by changes in the activity of electron transport chains in chloroplasts and mitochondria [3]. ROS act as secondary messengers mediating stress responses while their levels are tightly controlled to prevent cellular damage [4]. ROS scavenging comprises a large part of cellular responses to abiotic stresses. Scavengers include specific enzymes that reduce free radicals to water in multi-stage reactions [5], and anti-oxidative metabolites [6]. Proline (Pro) is a unique metabolite, which usually accumulates in response to different abiotic and biotic stresses, mostly due to stress-induced changes in the transcription of genes that encode enzymes of the evolutionary conserved glutamate (Glu)-proline-glutamate cycle [[7], [8], [9], [10], [11], [12]]. In mammals and plants, Pro catabolism to Glu occurs in mitochondria concomitantly delivering electrons to the mitochondrial electron transport chain (ETC). Intensive oxidation of Pro to P5C in mitochondria increases electron flux and elevates ROS generation [[8], [10]]. Although Pro accumulates in plant cells under stress conditions [[13], [14]], its protective role in overcoming stress damages is still under debate [[14], [15], [16], [17], [18]].
In a forward genetic screen, we isolated Arabidopsis thaliana T-DNA knockout mutants [19] capable of growing in the presence of lethal concentration of the Pro analogue L-thiazolidine-4-carboxylate (T4C, γ-thioproline) in NaCl-containing medium [8]. In Arabidopsis, salt-stress and dehydration-induced signaling elevate cellular Pro synthesis and simultaneously prevent mitochondrial Pro degradation by blocking the transcription of proline dehydrogenase 1 (ProDH1) leading to Pro accumulation [17]. Reduced ProDH1 activity during stress inhibits Pro oxidation to Δ(1)-pyrroline-5-carboxylate (P5C) and also T4C breakdown. T4C competes with Pro incorporation during translation causing protein disfunctioning, and blocking cell division and growth. Thus, T4C-tolerant mutants are expected to be impaired in normal stress-induced down-regulation of ProDH1 essential for Pro catabolism [8].
One of the T4C tolerant mutants, displaying higher tolerance to dehydration, carried a T-DNA insertion in the Arabidopsis gene AT5G13030 encoding a plant homologue of SELENOPROTEIN O (SELO). Selenoproteins contain selenocysteine (Sec) in their redox motifs [20]. Such substitution of sulfur atom with selenium in the cysteine residue increases nucleophilicity and redox activity. Conserved selenoprotein families with known functions include glutathione peroxidases [21], thioredoxin reductases [22] and deiodinases [23]. As plants lack selenocysteine-tRNA, their selenoprotein homologues contain cysteine residues instead of Sec in the redox motifs [[20], [24]]. Human SELO is located in mitochondria and possesses a C-terminal CxxSecSS redox motif, which catalyzes the formation of complexes through S-S and S-Se bridges with yet unknown protein partner(s), under oxidative conditions. Such redox-dependent complexes are also formed upon changing the motif to SxxSecSS, CxxCSS and SxxCSS [25], suggesting that the corresponding C-terminal CxxsCSS motif in plant SELO homologues likely maintains a similar redox activity. All members of the remarkably conserved SELO family contain a putative ATP-binding Walker-motive and some internal domains showing distant structural relationship to catalytic domains of porcine protein kinase A (PKA), including the archetypical His-Arg-Asp catalytic motif [26]. Nonetheless, the function of SELO, as well as its redox partner(s), remained so far unexplored.
Characterization of Arabidopsis selo mutation and its physiological effects in this study provides the first insight into the function of SELENOPROTEIN O in plants. SELO was localized to the chloroplast by using SELO-GFP fusion, and by its co-migration with thylakoid complexes on Blue-Native gels. Co-immunoprecipitation of proteins from isolated chloroplasts showed that SELO-GFP could interact with Ferredoxin NADP Reductase (FNR) and Protein Proton Gradient Regulation 5-Like 1A (PGRL1A), which are involved in electron transport from Photosystem I (PSI) [27], and also with the regulatory serine/threonine-protein kinase STN7. Lack of SELO in the selo mutant affected the redox poise, maintained in WT plants under normal growth conditions, by reducing H2O2 levels and enhancing expression of genes coding for ROS scavenging enzymes, mostly active in chloroplasts. A change in plant response to dehydration was also observed. The selo mutants were more tolerant to dehydration and their photosynthetic activity was extended compared with that of WT plants. Furthermore, regular signaling of Pro accumulation in response to stress was impaired in selo mutants leading to transcriptional induction, instead of silencing, of genes encoding enzymes of mitochondrial Pro catabolism during dehydration. To this end, our data indicate that SELENOPROTEIN O is involved in mediating ROS levels, mostly in chloroplasts and thereby affecting downstream signaling and stress responses.
Section snippets
Plant material, growth conditions, and stress induction
Arabidopsis thaliana, ecotype Columbia (Col-0), and its T-DNA insertion mutants were used in all experiments. The selo1-1 mutant was identified in the T-DNA-knockout mutant collection of Alvarado et al. [19], whereas the selo1-2 (GABI_956D07; [28]) and selo1-3 (SAIL_776_G08; [29]) mutants were obtained from the Arabidopsis Biological Resource Center (ABRC). Overexpression lines were generated using the ABRC cDNA stock GC105358 [30]. Plants were grown in growth chamber in pots under 16 h
Identification of selo knockout mutants and their physiological characterization
Using a collection of about 20,000 Arabidopsis T-DNA-tagged lines transformed with the promoter trap vector pTluc [19], we performed a forward genetic screen to identify mutants capable of growing in the presence of 3 mM T4C (Pro analogue) and 50 mM NaCl. We have previously demonstrated that salt-treatment inhibits the transcription of mitochondrial proline dehydrogenase (ProDH1), enhances Pro accumulation and ROS production, and prevents degradation of T4C that competes with Pro in the
Changes linked to SELO's absence
We identified plant SELO in a forward genetics approach designed to detect knockout mutants with disturbed abiotic stress signaling. Characterization of selo mutants demonstrated changes in ROS content and in transcript levels of nuclear encoded ROS scavenging enzymes most of them targeted to the chloroplast. Upon imposition of dehydration, selo mutants were more tolerant to the stress, and revealed disturbed Pro metabolism. The following sections are centered towards elucidating the linkage
In summary
The thylakoid-associated SELO has been studied by analyzing physiological events derived from its absence in the selo mutant. The recorded events include elevated dehydration tolerance with extended functioning of photosynthetic photosystems, and low levels of ROS under unstressed conditions linked to increased transcript levels of nuclear encoded ROS scavenging enzymes acting mostly in chloroplasts. Specific elevation of proline degradation occurs in mitochondria of selo mutant during
Accession numbers
AOX1A − AT3G22370; CAT2 − AT4G35090; CSD2 − AT2G28190; Cyclophilin − AT2G36130; FSD3 − AT5G23310; GR2 − AT3G54660; MDAR6 − AT1G63940; MSD1- AT3G10920; P5CDH − AT5G62530; P5CR − AT5G14800; P5CS1 − AT2G39800; P5CS2 – AT3G55610; PRODH1 − AT3G30775; PRODH2 − AT5G38710; PRXQ − AT3G26060; PTOX − AT4G22260; RBOHD − AT5G47910; sAPX − AT4G08390; SELO − AT5G13030; tAPX − AT1G77490; TRX-M2 − AT4G03520
Acknowledgements
We thank Jaideep Mathur (University of Guelph; Guelph, Canada) for the seeds of plants overexpressing tpFNR-YFP, Shimon Gepstein (The Technion; Haifa, Israel) for the anti RbcL antibodies and Anne Harzen (MPI for Plant Breeding Research; Cologne, Germany) for expert assistance in the mass spectrometry analyses. The advice and assistance of Haviva Eilenberg, Guilia Meshulam, Silvia Schuster and Chen Suchariano (Tel Aviv University, Tel Aviv, Israel) is greatly appreciated. We thank Dario Leister
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2021, Environmental and Experimental BotanyCitation Excerpt :We also detected an accumulation of tRNA ligase At5g27470, which may form selenocysteinyl-tRNA and participate in biosynthesis of selen-containing proteins. Interestingly, the set included Selenoprotein O (AT5G13030), which is reportedly involved in regulation of ROS levels and stress responses (Fichman et al., 2018). Its abundance was highest in cold-acclimated plants under L-PPFD, although the increase (1.4-fold compared to controls) did not meet the initial selection criteria.
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Authors with equal contribution.