Functional characterization of the DNA-binding protein from starved cells (DPS) as a molecular chaperone under heat stress.

The DNA-binding protein from starved cells, known as DPS, has been characterized as a crucial factor in protecting E. coli from external stresses. The DPS functions in various cellular processes, including protein-DNA binding, ferroxidase activity, compaction of chromosome and regulation of stress resistance gene expression. DPS proteins exist as oligomeric complexes; however, the specific biochemical activity of oligomeric DPS in conferring heat shock tolerance has not been fully understood. Therefore, we investigated the novel functional role of DPS under heat shock. To elucidate the functional role of DPS under heat shock conditions, we purified recombinant GST-DPS protein and demonstrated its thermostability and existence in its highly oligomeric form. Furthermore, we discovered that the hydrophobic region of GST-DPS influenced the formation of oligomers, which exhibited molecular chaperone activity, thereby preventing the aggregation of substrate proteins. Collectively, our findings highlight the novel functional role of DPS, as a molecular chaperone and may confer thermotolerance to E. coli.

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.

Functional Characterization of an Arabidopsis Profilin Protein as a Molecular Chaperone under Heat Shock Stress.

Profilins (PFNs) are actin monomer-binding proteins that function as antimicrobial agents in plant phloem sap. Although the roles of Arabidopsis thaliana profilin protein isoforms (AtPFNs) in regulating actin polymerization have already been described, their biochemical and molecular functions remain to be elucidated. Interestingly, a previous study indicated that AtPFN2 with high molecular weight (HMW) complexes showed lower antifungal activity than AtPFN1 with low molecular weight (LMW). These were bacterially expressed and purified to characterize the unknown functions of AtPFNs with different structures. In this study, we found that AtPFN1 and AtPFN2 proteins have LMW and HMW structures, respectively, but only AtPFN2 has a potential function as a molecular chaperone, which has never been reported elsewhere. AtPFN2 has better protein stability than AtPFN1 due to its higher molecular weight under heat shock conditions. The function of AtPFN2 as a holdase chaperone predominated in the HMW complexes, whereas the chaperone function of AtPFN1 was not observed in the LMW forms. These results suggest that AtPFN2 plays a critical role in plant tolerance by increasing hydrophobicity due to external heat stress.

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.

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.

HY5, a positive regulator of light signaling, negatively controls the unfolded protein response in Arabidopsis.

Light influences essentially all aspects of plant growth and development. Integration of light signaling with different stress response results in improvement of plant survival rates in ever changing environmental conditions. Diverse environmental stresses affect the protein-folding capacity of the endoplasmic reticulum (ER), thus evoking ER stress in plants. Consequently, the unfolded protein response (UPR), in which a set of molecular chaperones is expressed, is initiated in the ER to alleviate this stress. Although its underlying molecular mechanism remains unknown, light is believed to be required for the ER stress response. In this study, we demonstrate that increasing light intensity elevates the ER stress sensitivity of plants. Moreover, mutation of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to tolerance to ER stress. This enhanced tolerance of hy5 plants can be attributed to higher expression of UPR genes. HY5 negatively regulates the UPR by competing with basic leucine zipper 28 (bZIP28) to bind to the G-box-like element present in the ER stress response element (ERSE). Furthermore, we found that HY5 undergoes 26S proteasome-mediated degradation under ER stress conditions. Conclusively, we propose a molecular mechanism of crosstalk between the UPR and light signaling, mediated by HY5, which positively mediates light signaling, but negatively regulates UPR gene expression.

Redox-Dependent Structural Modification of Nucleoredoxin Triggers Defense Responses against Alternaria brassicicola in Arabidopsis.

In plants, thioredoxin (TRX) family proteins participate in various biological processes by regulating the oxidative stress response. However, their role in phytohormone signaling remains largely unknown. In this study, we investigated the functions of TRX proteins in Arabidopsis thaliana. Quantitative polymerase chain reaction (qPCR) experiments revealed that the expression of ARABIDOPSIS NUCLEOREDOXIN 1 (AtNRX1) is specifically induced by the application of jasmonic acid (JA) and upon inoculation with a necrotrophic fungal pathogen, Alternaria brassicicola. The AtNRX1 protein usually exists as a low molecular weight (LMW) monomer and functions as a reductase, but under oxidative stress AtNRX1 transforms into polymeric forms. However, the AtNRX1M3 mutant protein, harboring four cysteine-to-serine substitutions in the TRX domain, did not show structural modification under oxidative stress. The Arabidopsisatnrx1 null mutant showed greater resistance to A. brassicicola than wild-type plants. In addition, plants overexpressing both AtNRX1 and AtNRX1M3 were susceptible to A. brassicicola infection. Together, these findings suggest that AtNRX1 normally suppresses the expression of defense-responsive genes, as if it were a safety pin, but functions as a molecular sensor through its redox-dependent structural modification to induce disease resistance in plants.

Exploring Novel Functions of the Small GTPase Ypt1p under Heat-Shock by Characterizing a Temperature-Sensitive Mutant Yeast Strain, ypt1-G80D.

In our previous study, we found that Ypt1p, a Rab family small GTPase protein, exhibits a stress-driven structural and functional switch from a GTPase to a molecular chaperone, and mediates thermo tolerance in Saccharomyces cerevisiae. In the current study, we focused on the temperature-sensitive ypt1-G80D mutant, and found that the mutant cells are highly sensitive to heat-shock, due to a deficiency in the chaperone function of Ypt1pG80D. This defect results from an inability of the protein to form high molecular weight polymers, even though it retains almost normal GTPase function. The heat-stress sensitivity of ypt1-G80D cells was partially recovered by treatment with 4-phenylbutyric acid, a chemical chaperone. These findings indicate that loss of the chaperone function of Ypt1pG80D underlies the heat sensitivity of ypt1-G80D cells. We also compared the proteomes of YPT1 (wild-type) and ypt1-G80D cells to investigate Ypt1p-controlled proteins under heat-stress conditions. Our findings suggest that Ypt1p controls an abundance of proteins involved in metabolism, protein synthesis, cellular energy generation, stress response, and DNA regulation. Finally, we suggest that Ypt1p essentially regulates fundamental cellular processes under heat-stress conditions by acting as a molecular chaperone.

EMR, a cytosolic-abundant ring finger E3 ligase, mediates ER-associated protein degradation in Arabidopsis.

Investigation of the endoplasmic reticulum-associated degradation (ERAD) system in plants led to the identification of ERAD-mediating RING finger protein (EMR) as a plant-specific ERAD E3 ligase from Arabidopsis. EMR was significantly up-regulated under endoplasmic reticulum (ER) stress conditions. The EMR protein purified from bacteria displayed high E3 ligase activity, and tobacco leaf-produced EMR mediated mildew resistance locus O-12 (MLO12) degradation in a proteasome-dependent manner. Subcellular localization and coimmunoprecipitation analyses showed that EMR forms a complex with ubiquitin-conjugating enzyme 32 (UBC32) as a cytosolic interaction partner. Mutation of EMR and RNA interference (RNAi) increased the tolerance of plants to ER stress. EMR RNAi in the bri1-5 background led to partial recovery of the brassinosteroid (BR)-insensitive phenotypes as compared with the original mutant plants and increased ER stress tolerance. The presented results suggest that EMR is involved in the plant ERAD system that affects BR signaling under ER stress conditions as a novel Arabidopsis ring finger E3 ligase mainly present in cytosol while the previously identified ERAD E3 components are typically membrane-bound proteins.

The membrane-tethered NAC transcription factor, AtNTL7, contributes to ER-stress resistance in Arabidopsis.

We screened for endoplasmic reticulum (ER) stress-resistant mutants among 25 mutants of the Arabidopsis NTL (NAC with Transmembrane motif 1-Like) family. We identified a novel mutant, SALK_044777, showing strong resistance to ER stress. RT-PCR and genomic DNA sequence analyses identified the mutant as atntl7, which harbors a T-DNA insertion in the fourth exon of AtNTL7. Two other atntl7-mutant alleles, in which T-DNA was inserted in the second exon and third intron of AtNTL7, respectively, showed ER-stress sensitive phenotypes, suggesting that SALK_044777 is a gain-of-function mutant. Arabidopsis plants overexpressing AtNTL7 showed strong ER-stress resistance. Our findings suggest that AtNTL7 fragment is cleaved from the ER membrane under ER stress and translocates into the nucleus to induce downstream ER-stress responsive genes.

AtSRP1, SMALL RUBBER PARTICLE PROTEIN HOMOLOG, functions in pollen growth and development in Arabidopsis.

To identify novel roles of SMALL RUBBER PARTICLE PROTEIN Homolog in the non-rubber-producing plant Arabidopsis (AtSRP1), we isolated a T-DNA-insertion knock-out mutant (FLAG_543A05) and investigated its functional characteristics. AtSRP1 is predominantly expressed in reproductive organs and is localized to lipid droplets and ER. Compared to wild-type (WT) Arabidopsis, atsrp1 plants contain small siliques with a reduced number of heterogeneously shaped seeds. The size of anther and pollen grains in atsrp1 is highly irregular, with a lower grain number than WT. Therefore, AtSRP1 plays a novel role related to pollen growth and development in a non-rubber-producing plant.

Ribosomal P3 protein AtP3B of Arabidopsis acts as both protein and RNA chaperone to increase tolerance of heat and cold stresses.

The P3 proteins are plant-specific ribosomal P-proteins; however, their molecular functions have not been characterized. In a screen for components of heat-stable high-molecular weight (HMW) complexes, we isolated the P3 protein AtP3B from heat-treated Arabidopsis suspension cultures. By size-exclusion chromatography (SEC), SDS-PAGE and native PAGE followed by immunoblotting with anti-AtP3B antibody, we showed that AtP3B was stably retained in HMW complexes following heat shock. The level of AtP3B mRNA increased in response to both high- and low-temperature stresses. Bacterially expressed recombinant AtP3B protein exhibited both protein and RNA chaperone activities. Knockdown of AtP3B by RNAi made plants sensitive to both high- and low-temperature stresses, whereas overexpression of AtP3B increased tolerance of both conditions. Together, our results suggest that AtP3B protects cells against both high- and low-temperature stresses. These findings provide novel insight into the molecular functions and in vivo roles of acidic ribosomal P-proteins, thereby expanding our knowledge of the protein production machinery.

Stress-driven structural and functional switching of Ypt1p from a GTPase to a molecular chaperone mediates thermo tolerance in Saccharomyces cerevisiae.

Guanosine triphosphatases (GTPases) function as molecular switches in signal transduction pathways that enable cells to respond to extracellular stimuli. Saccharomyces cerevisiae yeast protein two 1 protein (Ypt1p) is a monomeric small GTPase that is essential for endoplasmic reticulum-to-Golgi trafficking. By size-exclusion chromatography, SDS-PAGE, and native PAGE, followed by immunoblot analysis with an anti-Ypt1p antibody, we found that Ypt1p structurally changed from low-molecular-weight (LMW) forms to high-molecular-weight (HMW) complexes after heat shock. Based on our results, Ypt1p exhibited dual functions both as a GTPase and a molecular chaperone, and furthermore, heat shock induced a functional switch from that of a GTPase to a molecular chaperone driven by the structural change from LMW to HMW forms. Subsequently, we found, by using a galactose-inducible expression system, that conditional overexpression of YPT1 in yeast cells enhanced the thermotolerance of cells by increasing the survival rate at 55°C by ∼60%, compared with the control cells expressing YPT1 in the wild-type level. Altogether, our results suggest that Ypt1p is involved in the cellular protection process under heat stress conditions. Also, these findings provide new insight into the in vivo roles of small GTP-binding proteins and have an impact on research and the investigation of human diseases.

The influence of light quality, circadian rhythm, and photoperiod on the CBF-mediated freezing tolerance.

Low temperature adversely affects crop yields by restraining plant growth and productivity. Most temperate plants have the potential to increase their freezing tolerance upon exposure to low but nonfreezing temperatures, a process known as cold acclimation. Various physiological, molecular, and metabolic changes occur during cold acclimation, which suggests that the plant cold stress response is a complex, vital phenomenon that involves more than one pathway. The C-Repeat Binding Factor (CBF) pathway is the most important and well-studied cold regulatory pathway that imparts freezing tolerance to plants. The regulation of freezing tolerance involves the action of phytochromes, which play an important role in light-mediated signalling to activate cold-induced gene expression through the CBF pathway. Under normal temperature conditions, CBF expression is regulated by the circadian clock through the action of a central oscillator and also day length (photoperiod). The phytochrome and phytochrome interacting factor are involved in the repression of the CBF expression under long day (LD) conditions. Apart from the CBF regulon, a novel pathway involving the Z-box element also mediates the cold acclimation response in a light-dependent manner. This review provides insights into the progress of cold acclimation in relation to light quality, circadian regulation, and photoperiodic regulation and also explains the underlying molecular mechanisms of cold acclimation for introducing the engineering of economically important, cold-tolerant plants.

Nucleoredoxin gene SINRX1 negatively regulates tomato immunity by activating SA signaling pathway.

The tomato (Solanum lycopersicum) is widely consumed globally and renowned for its health benefits, including the reduction of cardiovascular disease and prostate cancer risk. However, tomato production faces significant challenges, particularly due to various biotic stresses such as fungi, bacteria, and viruses. To address this challenges, we employed the CRISPR/Cas9 system to modify the tomato NUCLEOREDOXIN (SlNRX) genes (SlNRX1 and SlNRX2) belonging to the nucleocytoplasmic THIOREDOXIN subfamily. CRISPR/Cas9-mediated mutations in SlNRX1 (slnrx1) plants exhibited resistance against bacterial leaf pathogen Pseudomonas syringae pv. maculicola (Psm) ES4326, as well as the fungal pathogen Alternaria brassicicola. However, the slnrx2 plants did not display resistance. Notably, the slnrx1 demonstrated elevated levels of endogenous salicylic acid (SA) and reduced levels of jasmonic acid after Psm infection, in comparison to both wild-type (WT) and slnrx2 plants. Furthermore, transcriptional analysis revealed that genes involved in SA biosynthesis, such as ISOCHORISMATE SYNTHASE 1 (SlICS1) and ENHANCED DISEASE SUSCEPTIBILITY 5 (SlEDS5), were upregulated in slnrx1 compared to WT plants. In addition, a key regulator of systemic acquired resistance, PATHOGENESIS-RELATED 1 (PR1), exhibited increased expression in slnrx1 compared to WT. These findings suggest that SlNRX1 acts as a negative regulator of plant immunity, facilitating infection by the Psm pathogen through interference with the phytohormone SA signaling pathway. Thus, targeted mutagenesis of SlNRX1 is a promising genetic means to enhance biotic stress resistance in crop breeding.

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.

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 [...].

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.

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.

Redox sensor QSOX1 regulates plant immunity by targeting GSNOR to modulate ROS generation.

Reactive oxygen signaling regulates numerous biological processes, including stress responses in plants. Redox sensors transduce reactive oxygen signals into cellular responses. Here, we present biochemical evidence that a plant quiescin sulfhydryl oxidase homolog (QSOX1) is a redox sensor that negatively regulates plant immunity against a bacterial pathogen. The expression level of QSOX1 is inversely correlated with pathogen-induced reactive oxygen species (ROS) accumulation. Interestingly, QSOX1 both senses and regulates ROS levels by interactingn with and mediating redox regulation of S-nitrosoglutathione reductase, which, consistent with previous findings, influences reactive nitrogen-mediated regulation of ROS generation. Collectively, our data indicate that QSOX1 is a redox sensor that negatively regulates plant immunity by linking reactive oxygen and reactive nitrogen signaling to limit ROS production.