S-nitrosylation switches the Arabidopsis redox sensor protein, QSOX1, from an oxidoreductase to a molecular chaperone under heat stress.

The Arabidopsis quiescin sulfhydryl oxidase 1 (QSOX1) thiol-based redox sensor has been identified as a negative regulator of plant immunity. Here, we have found that small molecular weight proteins of QSOX1 were converted to high molecular weight (HMW) complexes upon exposure to heat stress and that this was accompanied by a switch in QSOX1 function from a thiol-reductase to a molecular chaperone. Plant treatment with S-nitrosoglutathione (GSNO), which causes nitrosylation of cysteine residues (S-nitrosylation), but not with H2O2, induced HMW QSOX1 complexes. Thus, functional switching of QSOX1 is induced by GSNO treatment. Accordingly, simultaneous treatment of plants with heat shock and GSNO led to a significant increase in QSOX1 chaperone activity by increasing its oligomerization. Consequently, transgenic Arabidopsis overexpressing QSOX1 (QSOX1OE) showed strong resistance to heat shock, whereas qsox1 knockout plants exhibited high sensitivity to heat stress. Plant treatment with GSNO under heat stress conditions increased their resistance to heat shock. We conclude that S-nitrosylation allows the thiol-based redox sensor, QSOX1, to respond to various external stresses in multiple ways.

The physiological role of thiol-based redox sensors in plant defense signaling.

Plants have developed multilayered defense strategies to adapt and acclimate to the kaleidoscopic environmental changes that rapidly produce reactive oxygen species (ROS) and induce redox changes. Thiol-based redox sensors containing the redox-sensitive cysteine residues act as the central machinery in plant defense signaling. Here, we review recent research on thiol-based redox sensors in plants, which perceive the changes in intracellular H2 O2 levels and activate specific downstream defense signaling. The review mainly focuses on the molecular mechanism of how the thiol sensors recognize internal/external stresses and respond to them by demonstrating several instances, such as cold-, drought-, salinity-, and pathogen-resistant signaling pathways. Also, we introduce another novel complex system of thiol-based redox sensors operating through the liquid-liquid phase separation.

Correction: Lee et al. Demyristoylation of the Cytoplasmic Redox Protein Trx-h2 Is Critical for Inducing a Rapid Cold Stress Response in Plants. Antioxidants 2021, 10, 1287.

In the original publication [...].

Universal Stress Protein regulates the circadian rhythm of central oscillator genes in Arabidopsis.

Environmental stresses restrict plant growth and development and decrease crop yield. The circadian clock is associated with the ability of a plant to adapt to daily environmental fluctuations and the production and consumption of energy. Here, we investigated the role of Arabidopsis Universal Stress Protein (USP; At3g53990) in the circadian regulation of nuclear clock genes. The Arabidopsis usp knockout mutant line exhibited critically diminished circadian amplitude of the central oscillator CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) but enhanced the amplitude of TIMING OF CAB EXPRESSION 1 (TOC1). However, the expression of USP under the control of its own promoter restored the circadian timing of both genes, suggesting that USP regulates the circadian rhythm of Arabidopsis central clock genes, CCA1 and TOC1.

Redox-mediated structural and functional switching of C-repeat binding factors enhances plant cold tolerance.

C-repeat binding factors (CBFs) are key cold-responsive transcription factors that play pleiotropic roles in the cold acclimation, growth, and development of plants. Cold-sensitive cbf knockout mutants and cold-tolerant CBF overexpression lines exhibit abnormal phenotypes at warm temperatures, suggesting that CBF activity is precisely regulated, and a critical threshold level must be maintained for proper plant growth under normal conditions. Cold-inducible CBFs also exist in warm-climate plants but as inactive disulfide-bonded oligomers. However, upon translocation to the nucleus under a cold snap, the h2-isotype of cytosolic thioredoxin (Trx-h2), reduces the oxidized (inactive) CBF oligomers and the newly synthesized CBF monomers, thus producing reduced (active) CBF monomers. Thus, the redox-dependent structural switching and functional activation of CBFs protect plants under cold stress.

Constitutive Photomorphogenic 1 Enhances ER Stress Tolerance in Arabidopsis.

Interaction between light signaling and stress response has been recently reported in plants. Here, we investigated the role of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a key regulator of light signaling, in endoplasmic reticulum (ER) stress response in Arabidopsis. The cop1-4 mutant Arabidopsis plants were highly sensitive to ER stress induced by treatment with tunicarmycin (Tm). Interestingly, the abundance of nuclear-localized COP1 increased under ER stress conditions. Complementation of cop1-4 mutant plants with the wild-type or variant types of COP1 revealed that the nuclear localization and dimerization of COP1 are essential for its function in plant ER stress response. Moreover, the protein amount of ELONGATED HYPOCOTYL 5 (HY5), which inhibits bZIP28 to activate the unfolded protein response (UPR), decreased under ER stress conditions in a COP1-dependent manner. Accordingly, the binding of bZIP28 to the BIP3 promoter was reduced in cop1-4 plants and increased in hy5 plants compared with the wild type. Furthermore, introduction of the hy5 mutant locus into the cop1-4 mutant background rescued its ER stress-sensitive phenotype. Altogether, our results suggest that COP1, a negative regulator of light signaling, positively controls ER stress response by partially degrading HY5 in the nucleus.

Demyristoylation of the Cytoplasmic Redox Protein Trx-h2 Is Critical for Inducing a Rapid Cold Stress Response in Plants.

In Arabidopsis, the cytosolic redox protein thioredoxin h2 (Trx-h2) is anchored to the cytoplasmic endomembrane through the myristoylated second glycine residue (Gly2). However, under cold stress, the cytosolic Trx-h2 is rapidly translocated to the nucleus, where it interacts with and reduces the cold-responsive C-repeat-binding factors (CBFs), thus activating cold-responsive (COR) genes. In this study, we investigated the significance of fatty acid modification of Trx-h2 under cold conditions by generating transgenic Arabidopsis lines in the trx-h2 mutant background, overexpressing Trx-h2 (Trx-h2OE/trx-h2) and its point mutation variant Trx-h2(G/A) [Trx-h2(G/A)OE/trx-h2], in which the Gly2 was replaced by alanine (Ala). Due to the lack of Gly2, Trx-h2(G/A) was incapable of myristoylation, and a part of Trx-h2(G/A) localized to the nucleus even under warm temperature. As no time is spent on the demyristoylation and subsequent nuclear translocation of Trx-h2(G/A) under a cold snap, the ability of Trx-h2(G/A) to protect plants from cold stress was greater than that of Trx-h2. Additionally, COR genes were up-regulated earlier in Trx-h2(G/A)2OE/trx-h2 plants than in Trx-h2OE/trx-h2 plants under cold stress. Consequently, Trx-h2(G/A)2OE/trx-h2 plants showed greater cold tolerance than Col-0 (wild type) and Trx-h2OE/trx-h2 plants. Overall, our results clearly demonstrate the significance of the demyristoylation of Trx-h2 in enhancing plant cold/freezing tolerance.

Disulfide reductase activity of thioredoxin-h2 imparts cold tolerance in Arabidopsis.

Many thioredoxin-h (Trx-h) proteins, cytosolic isotypes of Trxs, have been functionally characterized in plants; however, the physiological function of Arabidopsis Trx-h2, which harbors two active site cysteine (Cys) residues and an N-terminal extension peptide containing a fatty acid acylation site, remains unclear. In this study, we investigated the physiological function of Trx-h2 by performing several abiotic stress treatments using trx-h1-3 knockout mutant lines, and found that the reductase function of Trx-h2 is critical for cold resistance in Arabidopsis. Plants overexpressing Trx-h2 in the trx-h2 mutant background (Trx-h2OE/trx-h2) showed strong cold tolerant phenotypes compared with Col-0 (wild type) and trx-h2 mutant plants. By contrast, Trx-h2(C/S)OE/trx-h2 plants expressing a variant Trx-h2 protein, in which both active site Cys residues were substituted by serine (Ser) residues, showed high cold sensitivity, similar to trx-h2 plants. Moreover, cold-responsive (COR) genes were highly up-regulated in Trx-h2OE/trx-h2 plants but not in trx-h2 and Trx-h2(C/S)OE/trx-h2 plants under cold conditions. These results explicitly suggest that the cytosolic Trx-h2 protein relays the external cold stress signal to downstream cold defense signaling cascades through its protein disulfide reductase function.

Redox-dependent structural switch and CBF activation confer freezing tolerance in plants.

The activities of cold-responsive C-repeat-binding transcription factors (CBFs) are tightly controlled as they not only induce cold tolerance but also regulate normal plant growth under temperate conditions1-4. Thioredoxin h2 (Trx-h2)-a cytosolic redox protein identified as an interacting partner of CBF1-is normally anchored to cytoplasmic endomembranes through myristoylation at the second glycine residue5,6. However, after exposure to cold conditions, the demyristoylated Trx-h2 is translocated to the nucleus, where it reduces the oxidized (inactive) CBF oligomers and monomers. The reduced (active) monomers activate cold-regulated gene expression. Thus, in contrast to the Arabidopsis trx-h2 (AT5G39950) null mutant, Trx-h2 overexpression lines are highly cold tolerant. Our findings reveal the mechanism by which cold-mediated redox changes induce the structural switching and functional activation of CBFs, therefore conferring plant cold tolerance.