The E3 ubiquitin ligase COP1 regulates salt tolerance via GIGANTEA degradation in roots.

Excess soil salinity significantly impairs plant growth and development. Our previous reports demonstrated that the core circadian clock oscillator GIGANTEA (GI) negatively regulates salt stress tolerance by sequestering the SALT OVERLY SENSITIVE (SOS) 2 kinase, an essential component of the SOS pathway. Salt stress induces calcium-dependent cytoplasmic GI degradation, resulting in activation of the SOS pathway; however, the precise molecular mechanism governing GI degradation during salt stress remains enigmatic. Here, we demonstrate that salt-induced calcium signals promote the cytoplasmic partitioning of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), leading to the 26S proteasome-dependent degradation of GI exclusively in the roots. Salt stress-induced calcium signals accelerate the cytoplasmic localization of COP1 in the root cells, which targets GI for 26S proteasomal degradation. Align with this, the interaction between COP1 and GI is only observed in the roots, not the shoots, under salt-stress conditions. Notably, the gi-201 cop1-4 double mutant shows an enhanced tolerance to salt stress similar to gi-201, indicating that GI is epistatic to COP1 under salt-stress conditions. Taken together, our study provides critical insights into the molecular mechanisms governing the COP1-mediated proteasomal degradation of GI for salt stress tolerance, raising new possibilities for developing salt-tolerant crops.

OsGADD45a1: a multifaceted regulator of rice architecture, grain yield, and blast resistance.

KEY MESSAGE: The homolog gene of the Growth Arrest and DNA Damage-inducible 45 (GADD45) in rice functions in the regulation of plant architecture, grain yield, and blast resistance. The Growth Arrest and DNA Damage-inducible 45 (GADD45) family proteins, well-established stress sensors and tumor suppressors in mammals, serve as pivotal regulators of genotoxic stress responses and tumorigenesis. In contrast, the homolog and role of GADD45 in plants have remained unclear. Herein, using forward genetics, we identified an activation tagging mutant AC13 exhibited dwarf characteristics resulting from the loss-of-function of the rice GADD45α homolog, denoted as OsGADD45a1. osgadd45a1 mutants displayed reduced plant height, shortened panicle length, and decreased grain yield compared to the wild-type Kitaake. Conversely, no obvious differences in plant height, panicle length, or grain yield were observed between wild-type and OsGADD45a1 overexpression plants. OsGADD45a1 displayed relatively high expression in germinated seeds and panicles, with localization in both the nucleus and cytoplasm. RNA-sequencing analysis suggested a potential role for OsGADD45a1 in the regulation of photosynthesis, and binding partner identification indicates OsGADD45a1 interacts with OsRML1 to regulate rice growth. Intriguingly, our study unveiled a novel role for OsGADD45a1 in rice blast resistance, as osgadd45a1 mutant showed enhanced resistance to Magnaporthe oryzae, and the expression of OsGADD45a1 was diminished upon blast fungus treatment. The involvement of OsGADD45a1 in rice blast fungus resistance presents a groundbreaking finding. In summary, our results shed light on the multifaceted role of OsGADD45a1 in rice, encompassing biotic stress response and the modulation of several agricultural traits, including plant height, panicle length, and grain yield.

Inverse regulation of SOS1 and HKT1 protein localization and stability by SOS3/CBL4 in Arabidopsis thaliana.

To control net sodium (Na+) uptake, Arabidopsis plants utilize the plasma membrane (PM) Na+/H+ antiporter SOS1 to achieve Na+ efflux at the root and Na+ loading into the xylem, and the channel-like HKT1;1 protein that mediates the reverse flux of Na+ unloading off the xylem. Together, these opposing transport systems govern the partition of Na+ within the plant yet they must be finely co-regulated to prevent a futile cycle of xylem loading and unloading. Here, we show that the Arabidopsis SOS3 protein acts as the molecular switch governing these Na+ fluxes by favoring the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 kinase complex under salt stress, while commanding HKT1;1 protein degradation upon acute sodic stress. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1. These results indicate that roots first retain moderate amounts of salts to facilitate osmoregulation, yet when sodicity exceeds a set point, SOS3-dependent HKT1;1 degradation switches the balance toward Na+ export out of the root. Thus, SOS3 functionally links and co-regulates the two major Na+ transport systems operating in vascular plants controlling plant tolerance to salinity.

ABA INSENSITIVE 2 promotes flowering by inhibiting OST1/ABI5-dependent FLOWERING LOCUS C transcription in Arabidopsis.

The plant hormone abscisic acid (ABA) is an important regulator of plant growth and development and plays a crucial role in both biotic and abiotic stress responses. ABA modulates flowering time, but the precise molecular mechanism remains poorly understood. Here we report that ABA INSENSITIVE 2 (ABI2) is the only phosphatase from the ABA-signaling core that positively regulates the transition to flowering in Arabidopsis. Loss-of-function abi2-2 mutant shows significantly delayed flowering both under long day and short day conditions. Expression of floral repressor genes such as FLOWERING LOCUS C (FLC) and CYCLING DOF FACTOR 1 (CDF1) was significantly up-regulated in abi2-2 plants while expression of the flowering promoting genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was down-regulated. Through genetic interactions we further found that ost1-3 and abi5-1 mutations are epistatic to abi2-2, as both of them individually rescued the late flowering phenotype of abi2-2. Interestingly, phosphorylation and protein stability of ABA INSENSITIVE 5 (ABI5) were enhanced in abi2-2 plants suggesting that ABI2 dephosphorylates ABI5, thereby reducing protein stability and the capacity to induce FLC expression. Our findings uncovered the unexpected role of ABI2 in promoting flowering by inhibiting ABI5-mediated FLC expression in Arabidopsis.

A Mitochondrial Localized Chaperone Regulator OsBAG6 Functions in Saline-Alkaline Stress Tolerance in Rice.

B-cell lymphoma 2 (Bcl-2)-associated athanogene (BAG) family genes play prominent roles in regulating plant growth, development, and stress response. Although the molecular mechanism underlying BAG's response to abiotic stress has been studied in Arabidopsis, the function of OsBAG underlying saline-alkaline stress tolerance in rice remains unclear. In this study, OsBAG6, a chaperone regulator localized to mitochondria, was identified as a novel negative regulator of saline-alkaline stress tolerance in rice. The expression level of OsBAG6 was induced by high concentration of salt, high pH, heat and abscisic acid treatments. Overexpression of OsBAG6 in rice resulted in significantly reduced plant heights, grain size, grain weight, as well as higher sensitivity to saline-alkaline stress. By contrast, the osbag6 loss-of-function mutants exhibited decreased sensitivity to saline-alkaline stress. The transcriptomic analysis uncovered differentially expressed genes related to the function of "response to oxidative stress", "defense response", and "secondary metabolite biosynthetic process" in the shoots and roots of OsBAG6-overexpressing transgenic lines. Furthermore, cytoplasmic levels of Ca2+ increase rapidly in plants exposed to saline-alkaline stress. OsBAG6 bound to calcium sensor OsCaM1-1 under normal conditions, which was identified by comparative interactomics, but not in the presence of elevated Ca2+. Released OsCaM1-1 saturated with Ca2+ is then able to regulate downstream stress-responsive genes as part of the response to saline-alkaline stress. OsBAG6 also interacted with energy biosynthesis and metabolic pathway proteins that are involved in plant growth and saline-alkaline stress response mechanisms. This study reveals a novel function for mitochondrial localized OsBAG6 proteins in the saline-alkaline stress response alongside OsCaM1-1.

Plasma membrane-localized H+-ATPase OsAHA3 functions in saline-alkaline stress tolerance in rice.

KEY MESSAGE: A novel function of plasma membrane-localized H+-ATPase, OsAHA3, was identified in rice, which is involved in saline-alkaline tolerance and specifically responds to high pH during saline-alkaline stress. Saline-alkaline stress causes serious damage to crop production on irrigated land. Plants suffer more severe damage under saline-alkaline stress than under salinity stress alone. Plasma membrane-localized proton (H+) pump (H+-ATPase) is an important enzyme that controls plant growth and development by catalyzing H+ efflux and enabling effective charge balance. Many studies about the role of plasma membrane H+-ATPases in saline-alkaline stress tolerance have been reported in Arabidopsis, especially on the AtAHA2 (Arabidopsis thaliana H+-ATPase 2) gene; however, whether and how plasma membrane H+-ATPases play a role in saline-alkaline stress tolerance in rice remain unknown. Here, using the activation-tagged rice mutant pool, we found that the plasma membrane-localized H+-ATPase OsAHA3 (Oryza sativa autoinhibited H+-ATPase 3) is involved in saline-alkaline stress tolerance. Activation-tagged line 29 (AC29) was identified as a loss-of-function mutant of OsAHA3 and showed more severe growth retardation under saline-alkaline stress with high pH than under salinity stress. Moreover, osaha3 loss-of-function mutants generated by CRISPR/Cas9 system exhibited saline-alkaline stress sensitive phenotypes; staining of leaves with nitrotetrazolium blue chloride (NBT) and diaminobenzidine (DAB) revealed more reactive oxygen species (ROS) accumulation in osaha3 mutants. OsAHA3-overexpressing plants showed increased saline-alkaline stress tolerance than wild-type plants. Tissue-specific expression analysis revealed high expression level of OsAHA3 in leaf, sheath, glume, and panicle. Overall, our results revealed a novel function of plasma membrane-localized H+-ATPase, OsAHA3, which is involved in saline-alkaline stress tolerance and specifically responds to high pH.

Autophagy receptor OsNBR1 modulates salt stress tolerance in rice.

KEY MESSAGE: Autophagy receptor OsNBR1 modulates salt stress tolerance by affecting ROS accumulation in rice. The NBR1 (next to BRCA1 gene 1), as important selective receptors, whose functions have been reported in animals and plants. Although the function of NBR1 responses to abiotic stress has mostly been investigated in Arabidopsis thaliana, the role of NBR1 under salt stress conditions remains unclear in rice (Oryza sativa). In this study, by screening the previously generated activation-tagged line, we identified a mutant, activation tagging 10 (AC10), which exhibited salt stress-sensitive phenotypes. TAIL-PCR (thermal asymmetric interlaced PCR) showed that the AC10 line carried a loss-of-function mutation in the OsNBR1 gene. OsNBR1 was found to be a positive regulator of salt stress tolerance and was localized in aggregates. A loss-of-function mutation in OsNBR1 increased salt stress sensitivity, whereas overexpression of OsNBR1 enhanced salt stress resistance. The osnbr1 mutants showed higher ROS (reactive oxygen species) production, whereas the OsNBR1 overexpression (OsNBR1OE) lines showed lower ROS production, than Kitaake plants under normal and salt stress conditions. Furthermore, RNA-seq analysis revealed that expression of OsRBOH9 (respiratory burst oxidase homologue) was increased in osnbr1 mutants, resulting in increased ROS accumulation in osnbr1 mutants. Together our results established that OsNBR1 responds to salt stress by influencing accumulation of ROS rather than by regulating transport of Na+ and K+ in rice.

Type-B response regulator OsRR22 forms a transcriptional activation complex with OsSLR1 to modulate OsHKT2;1 expression in rice.

Soil salinity severely limits crop yields and quality. Plants have evolved several strategies to mitigate the adverse effects of salinity, including redistribution and compartmentalization of toxic ions using ion-specific transporters. However, the mechanisms underlying the regulation of these ion transporters have not been fully elucidated. Loss-of-function mutants of OsHKT2;1, which is involved in sodium uptake, exhibit strong salt stress-resistant phenotypes. In this study, OsHKT2;1 was identified as a transcriptional target of the type-B response regulator OsRR22. Loss-of-function osrr22 mutants showed resilience to salt stress, and OsRR22-overexpression plants were sensitive to salt stress. OsRR22 was found to activate the expression of OsHKT2;1 by directly binding to the promoter region of OsHKT2;1 via a consensus cis-element of type-B response regulators. Moreover, rice DELLA protein OsSLR1 directly interacted with OsRR22 and functioned as a transcriptional co-activator. This study has uncovered a novel transcriptional regulatory mechanism by which a type-B response regulator controls sodium transport under salinity stress.

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.

The HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE15-HISTONE DEACETYLASE9 complex associates with HYPONASTIC LEAVES 1 to modulate microRNA expression in response to abscisic acid signaling.

The regulation of microRNA (miRNA) biogenesis is crucial for maintaining plant homeostasis under biotic and abiotic stress. The crosstalk between the RNA polymerase II (Pol-II) complex and the miRNA processing machinery has emerged as a central hub modulating transcription and cotranscriptional processing of primary miRNA transcripts (pri-miRNAs). However, it remains unclear how miRNA-specific transcriptional regulators recognize MIRNA loci. Here, we show that the Arabidopsis (Arabidopsis thaliana) HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE15 (HOS15)-HISTONE DEACETYLASE9 (HDA9) complex is a conditional suppressor of miRNA biogenesis, particularly in response to abscisic acid (ABA). When treated with ABA, hos15/hda9 mutants show enhanced transcription of pri-miRNAs that is accompanied by increased processing, leading to overaccumulation of a set of mature miRNAs. Moreover, upon recognition of the nascent pri-miRNAs, the ABA-induced recruitment of the HOS15-HDA9 complex to MIRNA loci is guided by HYPONASTIC LEAVES 1 (HYL1). The HYL1-dependent recruitment of the HOS15-HDA9 complex to MIRNA loci suppresses expression of MIRNAs and processing of pri-miRNA. Most importantly, our findings indicate that nascent pri-miRNAs serve as scaffolds for recruiting transcriptional regulators, specifically to MIRNA loci. This indicates that RNA molecules can act as regulators of their own expression by causing a negative feedback loop that turns off their transcription, providing a self-buffering system.

Revisiting plant salt tolerance: novel components of the SOS pathway.

The Salt Overly Sensitive (SOS) pathway plays a central role in plant salinity tolerance. Since the discovery of the SOS pathway, transcriptional and post-translational regulations of its core components have garnered considerable attention. To date, several proteins that regulate these core components, either positively or negatively at the protein and transcript levels, have been identified. Here, we review recent advances in the understanding of the functional regulation of the core proteins of the SOS pathway and an expanding spectrum of their upstream effectors in plants. Furthermore, we also discuss how these novel regulators act as key signaling nodes of multilayer control of plant development and stress adaptation through modulation of the SOS core proteins at the transcriptional and post-translational levels.

HOS15 represses flowering by promoting GIGANTEA degradation in response to low temperature in Arabidopsis.

Flowering is the primary stage of the plant developmental transition and is tightly regulated by environmental factors such as light and temperature. However, the mechanisms by which temperature signals are integrated into the photoperiodic flowering pathway are still poorly understood. Here, we demonstrate that HOS15, which is known as a GI transcriptional repressor in the photoperiodic flowering pathway, controls flowering time in response to low ambient temperature. At 16°C, the hos15 mutant exhibits an early flowering phenotype, and HOS15 acts upstream of photoperiodic flowering genes (GI, CO, and FT). GI protein abundance is increased in the hos15 mutant and is insensitive to the proteasome inhibitor MG132. Furthermore, the hos15 mutant has a defect in low ambient temperature-mediated GI degradation, and HOS15 interacts with COP1, an E3 ubiquitin ligase for GI degradation. Phenotypic analyses of the hos15 cop1 double mutant revealed that repression of flowering by HOS15 is dependent on COP1 at 16°C. However, the HOS15-COP1 interaction was attenuated at 16°C, and GI protein abundance was additively increased in the hos15 cop1 double mutant, indicating that HOS15 acts independently of COP1 in GI turnover at low ambient temperature. This study proposes that HOS15 controls GI abundance through multiple modes as an E3 ubiquitin ligase and transcriptional repressor to coordinate appropriate flowering time in response to ambient environmental conditions such as temperature and day length.

GIGANTEA-ENHANCED EM LEVEL complex initiates drought escape response via dual function of ABA synthesis and flowering promotion.

Plants use the regulation of their circadian clock to adapt to daily environmental challenges, particularly water scarcity. During drought, plants accelerate flowering through a process called drought escape (DE) response, which is promoted by the circadian clock component GIGANTEA (GI). GI up-regulates the flowering inducer gene FLOWERING LOCUS T (FT). Phytohormone Abscisic acid (ABA) is also required for drought escape, and both GIGANTEA and Abscisic acid are interdependent in the transition. Recent research has revealed a new mechanism by which GIGANTEA and the protein ENHANCED EM LEVEL form a heterodimer complex that turns on ABA biosynthesis during drought stress by regulating the transcription of 9-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3). This highlights the close connection between the circadian clock and ABA regulation and reveals a new adaptive strategy for plants to cope with drought and initiates the DE response.

Plasma membrane-localized Hsp40/DNAJ chaperone protein facilitates OsSUVH7-OsBAG4-OsMYB106 transcriptional complex formation for OsHKT1;5 activation.

The salinization of irrigated land affects agricultural productivity. HIGH-AFFINITY POTASSIUM (K+ ) TRANSPORTER 1;5 (OsHKT1;5)-dependent sodium (Na+ ) transport is a key salt tolerance mechanism during rice growth and development. Using a previously generated high-throughput activation tagging-based T-DNA insertion mutant pool, we isolated a mutant exhibiting salt stress-sensitive phenotype, caused by a reduction in OsHKT1;5 transcripts. The salt stress-sensitive phenotype of this mutant results from the loss of function of OsDNAJ15, which encodes plasma membrane-localized heat shock protein 40 (Hsp40). osdnaj15 loss-of-function mutants show decreased plant height, increased leaf angle, and reduced grain number caused by shorter panicle length and fewer branches. On the other h'and, OsDNAJ15-overexpression plants showed salt stress-tolerant phenotypes. Intriguingly, salt stress facilitates the nuclear relocation of OsDNAJ15 so that it can interact with OsBAG4, and OsDNAJ15 and OsBAG4 synergistically facilitate the DNA-binding activity of OsMYB106 to positively regulate the expression of OsHKT1;5. Overall, our results reveal a novel function of plasma membrane-localized Hsp40 protein in modulating, alongside chaperon regulator OsBAG4, transcriptional regulation under salinity stress tolerance.

S-acylated and nucleus-localized SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 stabilizes GIGANTEA to regulate Arabidopsis flowering time under salt stress.

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering.

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 [...].

The thiol-reductase activity of YUCCA6 enhances nickel heavy metal stress tolerance in Arabidopsis.

Anthropogenic activities cause the leaching of heavy metals into groundwater and their accumulation in soil. Excess levels of heavy metals cause toxicity in plants, inducing the production of reactive oxygen species (ROS) and possible death caused by the resulting oxidative stress. Heavy metal stresses repress auxin biosynthesis and transport, inhibiting plant growth. Here, we investigated whether nickel (Ni) heavy metal toxicity is reduced by exogenous auxin application and whether Ni stress tolerance in Arabidopsis thaliana is mediated by the bifunctional enzyme YUCCA6 (YUC6), which functions as an auxin biosynthetic enzyme and a thiol-reductase (TR). We found that an application of up to 1 µM exogenous indole-3-acetic acid (IAA) reduces Ni stress toxicity. yuc6-1D, a dominant mutant of YUC6 with high auxin levels, was more tolerant of Ni stress than wild-type (WT) plants, despite absorbing significantly more Ni. Treatments of WT plants with YUCASIN, a specific inhibitor of YUC-mediated auxin biosynthesis, increased Ni toxicity; however yuc6-1D was not affected by YUCASIN and remained tolerant of Ni stress. This suggests that rather than the elevated IAA levels in yuc6-1D, the TR activity of YUC6 might be critical for Ni stress tolerance. The loss of TR activity in YUC6 caused by the point-mutation of Cys85 abolished the YUC6-mediated Ni stress tolerance. We also found that the Ni stress-induced ROS accumulation was inhibited in yuc6-1D plants, which consequently also showed reduced oxidative damage. An enzymatic assay and transcriptional analysis revealed that the peroxidase activity and transcription of PEROXIREDOXIN Q were enhanced by Ni stress to a greater level in yuc6-1D than in the WT. These findings imply that despite the need to maintain endogenous IAA levels for basal Ni stress tolerance, the TR activity of YUC6, not the elevated IAA levels, plays the predominant role inNi stress tolerance by lowering Ni-induced oxidative stress.

The Na+/H+ antiporter SALT OVERLY SENSITIVE 1 regulates salt compensation of circadian rhythms by stabilizing GIGANTEA in Arabidopsis.

The circadian clock is a timekeeping, homeostatic system that temporally coordinates all major cellular processes. The function of the circadian clock is compensated in the face of variable environmental conditions ranging from normal to stress-inducing conditions. Salinity is a critical environmental factor affecting plant growth, and plants have evolved the SALT OVERLY SENSITIVE (SOS) pathway to acquire halotolerance. However, the regulatory systems for clock compensation under salinity are unclear. Here, we show that the plasma membrane Na+/H+ antiporter SOS1 specifically functions as a salt-specific circadian clock regulator via GIGANTEA (GI) in Arabidopsis thaliana. SOS1 directly interacts with GI in a salt-dependent manner and stabilizes this protein to sustain a proper clock period under salinity conditions. SOS1 function in circadian clock regulation requires the salt-mediated secondary messengers cytosolic free calcium and reactive oxygen species, pointing to a distinct regulatory role for SOS1 in addition to its function as a transporter to maintain Na+ homeostasis. Our results demonstrate that SOS1 maintains homeostasis of the salt response under high or daily fluctuating salt levels. These findings highlight the genetic capacity of the circadian clock to maintain timekeeping activity over a broad range of salinity levels.

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.