Introduction

The circadian clock is an internal timekeeper that is conserved in many organisms and is important for coordinating biological processes with the external environment1. Key inputs such as light and temperature are important for synchronizing time of day information2,3,4. The clock consists of interlocked transcription-translation feedback loops (TTFLs) that regulate numerous cellular and biological processes5,6,7,8. In Arabidopsis thaliana (Arabidopsis), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), are two homologous MYB-like transcription factors expressed at dawn. Together, CCA1 and LHY transcriptionally repress the evening expressed TIMING OF CAB EXPRESSION1 (TOC1) by binding to the cis-regulatory motif, the evening element (EE)9. In turn, TOC1 negatively regulates the expression of CCA1 and LHY9,10. Outside of the core loop, CCA1 and LHY repress the expression of PSEUDORESPONSE REGULATORS (PRRs), PRR7, and PRR9, which are sequentially expressed throughout the day as part of the morning loop11,12. CCA1 and LHY also repress evening-expressed genes such as LUX, ELF3, and ELF4, which make up the evening complex (EC)13,14. Together, CCA1, LHY, the PRRs, the EC, and several additional components are important for maintaining a ~ 24-h period5,6,15,16.

In mammals, Circadian Locomotor Output Cycles Kaput (CLOCK) and Brain and Muscle ARNT-Like 1 (BMAL1) heterodimerize to make up the positive arm of the clock17. CLOCK-BMAL1 dimers activate the expression of Period (Per) and Cryptochrome (Cry) genes18. PER1/2 and CRY1/2 act as the negative arm of this feedback loop, repressing CLOCK and BMAL119. Outside of the main negative feedback loop, REV-ERBs and retinoic acid receptor (RAR)-related orphan receptor (ROR) (−α, −β, and −γ) repress and activate expression of BMAL1, respectively20,21.

In fungi (Neurospora crassa) the clock TTFL begins at dawn with the activation of White Collar-1 (WC-1) and White Collar-2 (WC-2), which make up the White Collar Complex (WCC)22. The WCC drives the expression of frequency (frq) which encodes FREQUENCY (FRQ)22,23. FRQ binds to FREQUENCY interacting RNA helicase (FRH), and this initiates its repressive role in the TTFL24,25. The self-interaction of FRQ along with FRH (FFC) and CK1 is important for FRQ stability and phosphorylation26,27,28. The FFC also interacts with the WCC, repressing its transcriptional activity by phosphorylating the WCC and inhibiting FRQ production29. As FRQ becomes hyperphosphorylated, the FFC can no longer inhibit WCC activity29. It is then ubiquitinated and degraded, which permits the WCC to reactivate the frq promoter and initiate a new cycle29. Despite the lack of sequence conservation of clock proteins across animals, fungi, and plants, aspects of their transcriptional-translational feedback and post-translational mechanisms are shared across kingdoms30,31,32,33.

In plants, the clock regulates various physiological, metabolic, and developmental processes, including hypocotyl elongation, flowering, and various stress responses34. At the molecular level, ~30% and 40% of the genes exhibit circadian oscillations at the transcriptional and translational levels, respectively35,36. The clock also shapes the Arabidopsis transcriptome through post-transcriptional processes such as alternative splicing and polyadenylation37,38. In animals and fungi, the circadian clock employs similar post-transcriptional mechanisms to regulate rhythmic gene expression and mRNA stability39,40,41,42.

Although the clock is known to play a role in regulating multiple layers of gene expression, the underlying mechanism coordinating different post-transcriptional processes in plants, specifically during heat stress, is still not well understood. Here, we explore the circadian regulation of mRNA localization into biomolecular condensates as a translational control mechanism during heat stress responses. Biomolecular condensates, including stress granules (SG), are dynamic membrane-less structures with a defined interfase that create distinct microenvironments that facilitate biological functions such as RNA metabolism and stress responses43. Drawing on studies in mammals and fungi that demonstrate a link between the circadian clock and cytoplasmic mRNA ribonucleoprotein granules (cmRNPgs), we discuss how the circadian clock may regulate SG formation and mRNA translation through post-transcriptional mechanisms. We hypothesize that biomolecular condensates serve as critical post-transcriptional mechanisms for mRNA regulation, potentially controlling circadian rhythms and stress responses in a time-of-day-dependent manner.

Post-transcriptional regulation of clock components

While post-translational control of clock proteins is integral to maintaining circadian rhythms, post-transcriptional regulation has also been shown to fine-tune the clock44,45. In Arabidopsis, several clock genes, including CCA1, LHY, and TOC1, undergo alternative splicing (AS), with intron retention being a prominent regulated event46. Fluctuations in temperature and light influence the AS of these clock genes, leading to the production of splice variants that are either targeted for Nonsense-Mediated RNA Decay (NMD) or translated into distinct isoforms46. For example, CCA1 undergoes an intron retention event under high light, which is reduced under low temperature46,47. The CCA1 splice variant, which retains intron 4, is predicted to encode a truncated protein, CCA1β, lacking its N-terminal MYB DNA-binding domain48. Under low temperatures, the suppression of CCA1 AS allows for the production of the functional full-length CCA1 (CCA1α), contributing to clock-mediated cold tolerance49. By contrast, splice variants of LHY, TOC1, PRR7, and PRR5 are targeted for NMD in response to low temperatures, thereby limiting the accumulation of their respective functional proteins50.

Temperature-dependent post-transcriptional regulation of clock genes has also been shown in mammals, fungi, and Drosophila51,52,53,54,55,56. For example, in Neurospora, frq undergoes temperature-dependent AS which generates the long (l) and short (s) isoforms of FRQ52,57. Higher ambient temperatures favor thermosensitive splicing of frq intron 6, leading to an increased l-FRQ to s-FRQ ratio52. This shift enhances the translation efficiency of FRQ at higher temperatures, revealing a thermosensitive mechanism that connects AS to translation regulation within the circadian clock53.

Notably, in Arabidopsis, altered expression in splicing factors such as PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) and SPLICEOSOMAL TIMEKEEPER LOCUS1 (STIPL1), can lead to circadian defects, underscoring the importance of AS in clock function58,59,60. In addition, the 5’-3’ mRNA decay pathway has been shown to play a role in clock function61. For example, mutations in SM-LIKE PROTEIN 1 (LSM1) and exoribonucleases 4 (XRN4), two components of the 5’ to 3’ mRNA decay pathway, resulted in long period phenotypes61. In Neurospora, the exosome, another mRNA decay pathway (3’ to 5’), is involved in regulating frq mRNA levels by interacting with the FFC62. Altogether, the circadian regulation of mRNA levels depends on the coordinated control of both mRNA synthesis and decay. These studies highlight the critical role of temperature-dependent post-transcriptional regulation in fine-tuning circadian clock function across diverse organisms, emphasizing its evolutionary importance in adapting to environmental changes.

Clock-controlled post-transcriptional regulation

In plants, the clock has been shown to regulate AS, a process that is partly attributed to the circadian-regulated splicing factor, AtSPF3037,38,47,63. Moreover, rhythmic alternative polyadenylation (APA) has also been observed as a result of clock regulation of APA-related genes, such as PAPS1 and PAPS237. Interestingly, genes associated with alternative splicing undergo rhythmic AS and APA emphasizing the intricate relationship between the circadian clock and AS. It is possible that the rhythmic regulation of AS-related genes ensures that the clock not only drives AS dynamics but may also rely on these processes for proper function. Studies going back to the 1990s have pointed to genes with arrhythmic transcription and rhythmic mRNA abundance and vice versa64,65,66,67. A recent study by Romanowski et al. revealed that ~21% of clock-controlled AS events are associated with arrhythmic transcripts38. In Arabidopsis, key clock-regulated genes such as CHLOROPHYLL A/B BINDING PROTEIN1 (CAB1) and CATALASE3 (CAT3) exhibit rhythmic transcription but reduced or non-rhythmic steady-state mRNA levels64,65,66,67. In addition, NITRASE REDUCTASE (NIA2) shows rhythmic mRNA accumulation in constant conditions, despite having little or no rhythmicity in transcription64,65,66,67. This phenomenon can be a result of clock-controlled mRNA stability.

In plants, the circadian clock has been shown to regulate mRNA decay rates in a time-of-day-dependent manner via the DST-mediated mRNA decay pathway68. Specifically, the half-life of CCR-LIKE (CCL) mRNA was found to be regulated by the time of day, and depend on the downstream (DST) element located in the 3’ untranslated region (UTR) of CCL68. More recently, CCL mRNA was shown to be sequestered into processing bodies (PBs) under heat stress69. This raises the question of whether the DST element mediates circadian mRNA sequestration into PBs to sustain robust mRNA oscillations. Whether the circadian clock can regulate mRNA localization patterns in response to heat stress remains an open question.

In mammals, post-transcriptional regulation also plays a significant role in shaping circadian rhythms70,71. Coupling genome-wide RNA polymerase II (RNAPII) profiling with whole transcriptome RNA sequencing revealed that ~22% of cycling transcripts depend on rhythmic transcription71. In addition, Nascent-Seq analysis of genome-wide transcriptional rhythms in the mouse liver reveals that ~70% of genes showing rhythmic mRNA levels lack strong rhythmic mRNA synthesis70,71. Together, these findings highlight the multifaceted role of the circadian clock in shaping rhythmic gene expression and maintaining circadian homeostasis through regulating RNA metabolism, from AS and APA to mRNA stability and decay. It would be particularly interesting to explore how environmental factors such as heat stress influence these processes, potentially uncovering additional layers of regulation and adaptation within the circadian network. These mechanistic insights are increasingly relevant in the context of climate change.

Translation regulation by the clock

By regulating RNA metabolism, the circadian clock fine-tunes translation, aligning protein synthesis with biological rhythms35,72,73,74,75. For example, in Arabidopsis, ribosome loading, the binding of ribosomes to mRNA, accumulates to ~70% during the day, reduces to 40% at the end of the night, and depletes to 20% during periods of starch exhaustion76. Plants overexpressing CCA1 (CCA1-OX) showed a 6-hour phase shift in translation and the rhythmic patterns of ribosome loading observed in wild type under continuous light were abolished72. Of these transcripts, CCL, which was mentioned above as having rhythmic mRNA half-life and sequestered into PBs, was shown to lose rhythmic ribosome loading in CCA1-OX. In addition, translating ribosome affinity purification sequencing (TRAP-seq) revealed that 40% of the translatome showed circadian oscillations under free-running conditions, suggesting that the clock and time of day regulate mRNA-ribosome associations35. In an analysis comparing cycling patterns in the transcriptome versus the translatome, we observed that ~36% of the transcripts are only rhythmic at the level of translation35 (Fig. 1A). This can be due to several post-transcriptional processes including rhythmicity in mRNA abundance due to clock-controlled AS, mRNA stability, or transcript sequestration into biomolecular condensates (Fig. 1B). In addition, this study revealed that in response to heat stress (37 °C for 1 h), the time of day also modulates the response of circadian TRAP mRNA abundance35. Notably, a subset of genes show heat stress induction at the transcriptional level but not at the level of translation35 (Fig. 1A). This observed discrepancy raises the intriguing possibility of sequestration of mRNAs into heat-induced biomolecular condensates such as stress granules (SGs), to modulate translation in a time-of-day dependent manner (Fig. 1B). SGs are dynamic biomolecular condensates that form in response to stress conditions, including heat, and are known to sequester specific mRNAs, translation initiation factors, and ribosomal subunits77. This sequestration halts active translation, potentially allowing cells to conserve resources or prioritize the translation of stress-responsive proteins.

Fig. 1: Post-transcriptional processes regulating circadian rhythmicity and heat stress responses.
figure 1

A Transcriptome vs translatome profiles of genes that show post-transcriptional regulation under ambient conditions (22 °C) or in response to heat stress (1 h 37 °C)35. Left side shows the profile of genes that have non-rhythmic steady-state mRNA accumulation but rhythmic mRNA translation in free-running conditions. The right side shows the profile of genes that are upregulated at the transcriptional level, but not at the translational level, during heat stress. ZT0 - 24 indicates continuous light conditions, ZT (Zeitgeber). White shading on the graph represents day and gray shading represents subjective night. B The circadian clock and/or time-of-day can modulate the post-transcriptional and translational response of genes by regulating (1) alternative splicing leading to nuclear retention of one of the transcripts, (2) mRNA decay via translation or by sequestration into p-bodies (PBs), and (3) mRNA sequestration into stress granules (SGs) under heat stress. Figure Created in BioRender. Brown, G and Nagel, D. (2025) https://BioRender.com/95nkiov.

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In Neurospora, the circadian clock modulates translation by regulating initiation through the eIF2α kinase Cross Pathway Control-3 (CPC-3)73 (Fig. 2A). During the day, CPC-3 phosphorylates and inactivates eIF2α, leading to a reduction in translation initiation78. At night, the clock regulates Protein Phosphatase-1 (PPP-1), the eIF2α phosphatase, enabling the dephosphorylation and activation of eIF2α, which promotes an increase in translation initiation74. Additionally, the clock has also been shown to modulate translation by regulating aminoacyl-tRNA synthetase levels, with peak levels occurring at night75. These two pieces of evidence suggest that in Neurospora, the clock coordinates protein synthesis to occur predominantly at night.

Fig. 2: The clock regulates cytoplasmic biomolecular condensates in animals and fungi.
figure 2

A The clock regulates rhythmicity of the kinase (GCN2/CPC-3) that phosphorylates eIF2α (P-eIF2α)78,115. In fungi, rhythmic eIF2α phosphorylation inhibits translation initiation of certain mRNAs in a time-of-day dependent manner78. Inhibition of translation initiation by P-eIF2α promotes the sequestration of specific transcripts into cmRNPgs, such as PBs, which results in rhythmic translation74. B In mice, reduction in expression of the clock gene, BMAL1, increases the levels of P-eIF2α and results in more stress granules during the subjective night under sodium arsenite (SA) stress114. ZT0 - 24 indicates continuous light conditions, ZT (Zeitgeber). White rectangle on the graph represents the day and the gray rectangle represents the subjective night. Figure created in BioRender. Brown, G and Nagel, D. (2025) https://BioRender.com/snbvhrq.

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However, plants are autotrophic and rely on photosynthesis, which generates ATP and NADPH primarily during the day79. Thus, the daytime is ideal for energy-intensive processes like protein synthesis because energy is abundantly available. Indeed, in Bonnot et al. the TRAP-specific cycling genes show phase enrichment during the day35. In a different Arabidopsis study, unanticipated darkness caused a 17% reduction in polysome levels consistent with inhibition of translation initiation80. These observations suggest that plants prioritize protein synthesis during the day to align with energy availability from photosynthesis. However, stress conditions, such as heat stress, demand rapid and efficient reprogramming of cellular processes to conserve resources and facilitate survival. We propose that under heat stress, plants may employ the formation and regulation of SGs, potentially under the influence of the circadian clock, to optimize energy use and enhance stress tolerance.

Biomolecular condensates in cellular stress responses

Cytoplasmic mRNA ribonucleoprotein condensates (cmRNPgs) such as SGs and PBs have emerged as essential players in RNA metabolism. While these biomolecular condensates can have distinct roles in mRNA regulation, both form through liquid-liquid phase separation and are involved in the sequestration of molecules such as proteins and nucleic acids at discrete cellular sites81,82. cmRNPgs are widely considered to play a role in organizing protein-RNA complexes to regulate translation by modulating mRNA protection and degradation during stress conditions and the subsequent recovery period83. SGs are formed in response in response to various environmental stresses and their formation is triggered by global translation stalling82.

In plants, SGs form in response stresses such as heat shock84 and hypoxia85 and they typically disassemble during the recovery phase, depending on the severity of the stress86. However, the underlying mechanism that drives SG assembly in plants remains unknown. Condensates are suggested to be triggered by a combination of site-specific and chemically specific interactions, facilitated in some cases by structured interfaces or sequence-based molecular recognition, often involving intrinsically disordered regions (IDRs)87,88,89,90,91,92,93,94. In plants, and other eukaryotes, clock proteins have been shown to contain IDRs and form biomolecular condensates as part of their regulatory function95,96,97,98,99. Additionally, a large number of the SG proteome is composed of intrinsically disordered proteins or proteins containing IDRs100. In Arabidopsis, SG-mass spectrometry analyses utilizing various SG markers, including Rbp47b, TSN2, and RGBD2/4, indicate a lack of significant homogeneity in the interactomes of these markers under heat stress conditions, with only three overlapping proteins101,102,103. Furthermore, the proteasome has been shown to localize in SGs under heat stress and this may contribute to SG clearance during the recovery phase86. More recently, it was found that autophagy proteins relocate to SGs under heat stress to suppress autophagy but are released during recovery into the cytosol, where autophagy is reinitiated, and heat-induced ubiquitinated insoluble protein aggregates are cleared104. In plants, SG-mass spectrometry has revealed ribosomal proteins and translation-related factors suggesting that mRNAs may also be translated in SGs as observed in mammalian cells101,105. However, evidence for active translation of SG-sequestered mRNAs has not been demonstrated in plants. This highlights a gap in knowledge, as most stress granule research has been conducted in mammalian and yeast cells, leaving the mechanisms and functions of stress granules in plants and in the context of the clock, largely unexplored.

Unlike stress granules, which form in response to stress, PBs are constitutively present in the cytoplasm. Both SGs and PBs sequester mRNAs and although the primary role of PBs is to facilitate mRNA decay, recent evidence suggests that mRNAs are not always degraded in PBs69,106,107. Animal and plant PBs house many components of the RNA decapping machinery such as DECAPPING1 (DCP1) and XRN4108. Recent studies revealed that the size of PBs correlates with their RNA degradation capacity69. For example, two distinct populations of PBs were identified, with larger and smaller PBs acting more as RNA storage and degrading structures, respectively69. Furthermore, in response to stress, PB size can increase, and the proteome of PBs becomes less similar to that of heat-induced SGs suggesting a distinct role for these two cytoplasmic condensates in heat stress responses109,110,111. Despite the well-documented roles of SGs and PBs in regulating stress responses in animals and fungi, in plants, the direct involvement of the circadian clock in these processes remains an open question.

Circadian regulation of biomolecular condensates

In plants, many genes exhibit rhythmic translation despite having arrhythmic gene expression patterns35. Additionally, several clock-controlled genes are induced by heat stress primarily at the transcriptional level, suggesting the involvement of post-transcriptional regulation35. One potential mechanism is the sequestration of mRNAs into cytoplasmic condensates, such as SGs and PBs, which can allow the circadian clock to regulate the timing and translation of these genes in a heat-stress-specific or time-of-day-dependent manner. In mammalian cells, the clock regulates PB formation via clock-controlled LSM1 expression, a necessary component for PB formation112,113. Additionally, alteration of the clock by silencing BMAL1 resulted in reduced PB formation112,113. The circadian clock has been shown to regulate sodium arsenite induced stress granules in animals114 (Fig. 2B). In mice, oscillations in BMAL1 expression are closely correlated with phosphorylation of eIF2α (P-eIF2α), with the lowest levels of BMAL1 expression coinciding with a peak in eIF2α phosphorylation114 (Fig. 2B). In mice, the clock regulates the kinase (GCN2) responsible for phosphorylation of eIF2α115. Phosphorylation of eIF2α results in translation inhibition, which can be associated with the sequestration of specific mRNAs into SGs during stress116,117,118. Once eIF2α is dephosphorylated, it returns to its active state, allowing translation to resume for certain mRNAs119. Moreover, alterations in BMAL1 expression have been shown to affect the formation and dynamics of stress granules, suggesting a direct role for the circadian clock in regulating translation through SGs in animals114.

Similar patterns have been observed in Neurospora, where the clock regulates cmRNPg formation via increased P-eIF2α levels during the subjective day74 (Fig. 2A). Translation inhibition by P-eIF2α results in the sequestration of certain transcripts into cmRNPgs and de- novo motif analysis of circadian translation-initiation-controlled (cTIC) genes suggests an enrichment for PB localization signals (Fig. 2A). Using the cTIC gene ZIP-1, it was demonstrated that clock regulated eIF2α activity governs the rhythmic translation of specific mRNAs by sequestering them into cmRNP granules, particularly PB74 (Fig. 2A).

In plants, specifically in Arabidopsis, the circadian clock likely regulates the formation and composition of SGs, though direct evidence linking the clock to SG dynamics in plants remains limited. However, the rhythmic expression patterns of SG-associated proteins, such as RGBD2/4 and GRP7, suggest that the clock may indeed influence SG formation and function35. Mass-spectrometry analysis of SG proteins under heat stress, including Rbp47b, RGBD2/4, and TSN2, reveals that approximately 60%, 80%, and 50% of the proteins associated with them, respectively, are transcriptionally regulated by the clock101,102,103. Furthermore, among the mRNAs associated with SGs through interactions with RGBD2/4 and ALKBH9B, 68% and 48% are clock-regulated, respectively103,120. For example, BBX16 and BBX17 mRNAs are associated with ALKBH9B under heat stress, and both show strong responsiveness to heat stress while displaying robust circadian rhythms35.

Additionally, several SG-associated proteins are linked to the circadian system. Rbp45b and RGBD2, for example, interact with the evening clock protein, TOC1121. GRP7 and CRB, two other SG-associated proteins, have been implicated in circadian regulation122,123,124,125. GRP7 regulates the steady-state levels and alternative splicing of multiple circadian transcripts, while mutants of CRB lead to altered expression of core clock genes. GRP7 also modulates the stability of LHCB1.1, a chlorophyll-binding protein, by regulating its mRNA half-life in a circadian-dependent manner. Interestingly, LHCB1.1 mRNA has been identified among the mRNAs associated with RGBD2/4 under heat stress103. While it is unclear whether GRP7 sequesters LHCB1.1 mRNA into SGs to control its translation during heat stress, GRP7 emerges as a compelling candidate for investigating the role of the circadian clock in SG formation and mRNA composition (Fig. 3A). Because the clock is known to coordinate energy availability and metabolism126, it raises the intriguing possibility that by modulating SG formation at specific times of day, the clock may optimize the plant’s responses to heat stress and other environmental challenges (Fig. 3B).

Fig. 3: Model depicting how the clock may regulate stress granules in Arabidopsis.
figure 3

A The clock regulates RNA-binding proteins, GRP7 and CRB35,124. GRP7 and CRB have been shown to help maintain circadian rhythms122,124,125. Under heat stress, GRP7 and CRB localize into RGBD2/4 associated SGs and may sequester specific transcripts in a time-of-day dependent manner103. This can lead to clock controlled mRNA sequestration into stress granules (SGs) and/or fine-tuning of translation under heat stress that is dependent on the clock. B The clock coordinates energy availability and metabolism. During times of low energy availability such as end of night and just prior to dawn the clock may promote storage of molecules into SGs during heat stress. This may lead to a reduction of translation and an increase in SG formation at night as a clock-controlled heat stress response mechanism. Figure created in BioRender. Brown, G and Nagel, D. (2025) https://BioRender.com/3ro4j36.

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Conclusion

It is clear that the regulatory mechanisms underlying circadian control of gene expression and cellular responses are complex. Given the negative impacts of changing weather patterns on crop growth and productivity, understanding the underlying regulatory mechanisms that may confer and optimize heat tolerance is necessary. In plants, SGs form in response to various environmental stresses, including heat, drought, and salinity84. However, the influence of the circadian clock on SG formation and dynamics in plants is not as well understood. Evidence from animals and fungi suggests that the clock may control SG formation by modulating key translation factors and RNA-binding proteins (RBPs) that regulate mRNA stability and translation. We hypothesized that the circadian clock in plants could regulate SG formation and function, potentially optimizing stress responses by modulating the timing of mRNA translation and stability. Sequestration into SG while acting as a cellular protective mechanism may be critical for rapid activation of recovery processes once a stress is removed. However, it is important to mention that the field of biomolecular condensates is still emerging and rapidly evolving, and as new discoveries are made, the hypotheses and models proposed in this review will need to be updated or reinterpreted to reflect the advancement in knowledge. As the impacts of climate change intensify, plants will increasingly depend on mechanisms that enhance stress tolerance and recovery. Unraveling the role of the circadian clock in stress granule dynamics presents a promising opportunity to improve heat stress resilience in plants.