LNKs-RVEs complex ticks in the circadian gating of plant temperature stress responses

Recently, Kidokoro et al. found that protein complex LNK3,4-RVE4,8 and LNK1,2-RVE4,8 of the circadian clock modulates plant cold- and high-temperature tolerance, respectively. Here, we reviewed the discovery of LNKs, the dynamically formed morning-phased clock complexes, and their critical role on endogenous circadian rhythms. In addition, we summarized the research work on LNKs with the interacting proteins RVEs, CCA1 in temperature responses and discussed how the circadian clock confer increased fitness via gating the rhythmic expression of their target genes.

The circadian clock is a cell-autonomous timekeeping mechanism that readily transduces cyclic changes in external cues (time giver or Zeitgebers) into rhythmic physiological and biochemical processes, thereby achieving the adaptation to photoperiod and ambient temperatures of living organisms.In the model plant Arabidopsis thaliana, Myb-like transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), REVEILLEs (RVEs), and PSEUDO-RESPONSE REGULATORs (PRR9, PRR7) transcripts and proteins are enriched in the daytime, while PRR5, PRR3, PRR1/TOC1 (TIMING OF CAB EXPRESSION1), ELF3-ELF4-LUX (known as Evening Complex, EC) reach their expression peaks in the evening or at night.These core oscillators expressed during the 24-h day constitute the multiple interlocked transcriptional-translational feedback loops (TTFLs) of the circadian clock.The 6% of (Xie et al. 2014).In addition, LNK1 and LNK2 also interact with RNA Polymerase II and the transcript elongation FACT complex to determine the RNA Pol II and H3K4me3 deposition at the PRR5 and TOC1 loci and target mRNA circadian oscillations (Ma et al. 2018).The LNK family has four members that share conserved protein sequences, of which LNK1 and LNK2 have a molecular weight of about 66 kDa, while LNK3 and LNK4 look like truncated proteins with a molecular weight of about 30 kDa.Unlike lnk1 and lnk2, the parameters of circadian rhythm in lnk3-1 and lnk4-1 were not altered under free-running conditions (constant light, 22 °C) (Xie et al. 2014).However, the lnk1 2 3 4 quadruple mutant (lnkQ) displayed a 1.3-h lengthened period of circadian rhythm than lnk1 lnk2 double mutant, and it was more significant in rosette size and biomass than the lnk1 lnk2 plant.(de Leone et al. 2019).Therefore, the function of LNK3 and LNK4 in the circadian clock system was still largely unknown.
Recently, Kidokoro et al. found that LNK3 and LNK4 activate the expression of the clock gene PRR5 at lower temperature (4 °C), and that the LNK3,4-RVE4,8 complex and the LNK1,2-RVE4,8 complex determine plant temperature stress tolerance by regulating the expression of cold-or heat-responsive genes, respectively (Kidokoro et al. 2023).In this study, PRR5 transcript level at 22 °C was significantly repressed in lnk1 lnk2 double mutant and was consistent in lnk3 lnk4 and wild-type (Col-0) plant, indicating that LNK1 and LNK2 as an activator is regulating PRR5 transcription as previously known.At chilling conditions (4 °C), LNK1 and LNK2 completely lost their regulation of PRR5 expression, as PRR5 transcript levels are the same as in the wild type.Of interest, cold stress contributes to the enrichment of PRR5 transcripts in lnk3 lnk4 by about 40% of that in wild type, suggesting that LNK3 and LNK4 show an activating effect on PRR5 expression in response to temperature changes.This finding reveals a temperature-dependent function of LNK3 and LNK4 in the circadian clock LNKs-PRR5 loop.Next, the circadian rhythms need to be measured to know whether LNK3, LNK4 and PRR5 determine the clock pace, amplitude, or phase in cold conditions.
This article spotlights the selective interaction of LNK1, LNK2 or LNK3, LNK4 with the circadian oscillator RVE4,8 and the response of LNKs-RVEs protein complexes to environmental cold or heat stress.The results showed that all four LNKs could be induced by low temperature signals (4 °C), while the LNK3 and LNK4 transcript levels were significantly higher than their expression at 22 °C, which was different from that of LNK1 and LNK2.The DREB1A/CBF3, DREB1B/ CBF1 and DREB1C/CBF2 in Arabidopsis are known to be transcription factors rapidly induced by cold and freezing treatment, and they enhance low temperature stress tolerance by activating the expression of cold response (COR) genes with promoter-containing CRT/ DRE cis-elements (Jaglo-Ottosen et al. 1998).Kidokoro et al. found that the cold-induced DREB1A expression was similar in wild-type and lnk1 lnk2 plants, whereas it was reduced by more than 50% in lnk3 lnk4 and lnk1 lnk2 lnk3 lnk4/lnkQua mutants.Previously, Kidokoro et al. revealed RVE4 and RVE8 protein accumulation in the nucleus in response to chilling/4 °C and that RVE4 and RVE8 protein binds more to DREB1A promoter to activate its expression under cold stress condition than CCA1 and LHY (Kidokoro et al. 2021).In the current article, the expression of cold-induced DREB1A was also reduced by 60-80% in rve4 rve8 mutant (Kidokoro et al. 2023).After deletion analysis of the C-and N-terminal regions of RVE8 protein, it was found that the C-terminal of RVE8 determined its regulation of DREB1A expression under cold stress.This study also confirmed protein interactions between LNK3,4 and RVE4,8 in the nuclei at both 24 °C and 4 °C conditions.Importantly, transcriptome sequencing of lnkQua and wild-type plants treated with 4 °C for 3 h was performed to help identify the LNKs-regulated signaling pathways and hundreds of target genes including COR15A and COR47.Further physiological phenotypic analysis of -10 °C treatment in 4 °C/cold-acclimated plants confirmed RVEs and LNKs as regulating cold-induced freezing tolerance.Another highlight of this study is that the authors found phosphorylation of LNK3 and LNK4 proteins and an increased amount of phosphorylated LNK3 and LNK4 after 3-h 4 °C treatment.In summary, this article identified the LNK3,4-RVEs module, which plays a critical role in COR gene expression and low-temperature tolerance of plants.
On the other hand, in this study, heat stress-induced ETHYLENE RESPONSIVE FACTORS 53/ERF53 and ERF54 gene expression was more repressed in lnk1 lnk2 than in lnk3 lnk4 plants in response to heat stress (37 °C), suggesting the major contribution of LNK1 and LNK2 in high temperature tolerance.Further in the analysis of 45-min 43 °C heat shock after 37 °C acclimation, lnk1 lnk2 plants displayed more tolerance than lnk3 lnk4 mutant.This result provides the working hypothesis that the LNK1,2-RVE4,8 complex may dominate the heat shock response (HSR), distinct from the role of LNK3,4-RVE4,8 in cold and freezing resistance (Fig. 1).
The protein-protein interactions between the circadian core oscillators show a 24-h rhythmicity, predicting that the temporal window of peak expression of the morning-phased or evening-phased protein complex are associated with the anticipation of fluctuations in environmental signals.This article revealed that protein complexes composed of LNK1,2 and LNK3,4 recruit RVEs with distinct sensitivity to temperature fluctuations; this finding contributes to the understanding of the molecular and physiological functions of transcriptional cofactors, including LNKs.It is also worth exploring the role of LNK1-4 in the transcriptional regulation of their target genes, as LNKs interact with transcriptional activators RVEs along with transcriptional repressors CCA1 and LHY, and these protein complexes are all enriched in the early morning.It is known that dysfunction of PRR9, PRR7, and COR27, COR28 enhances freezing tolerance (-7 °C) without cold assimilation; and PRR9 and PRR7 suppress CCA1 expression in TTFLs of the circadian clock (Wang et al. 2017).Cold stress attenuates CCA1 binding to the COR27 and COR28 promoter, which potentially be involved in the low temperature response in a CBF-independent pathway.Recently, Sorkin et al. identified the RVE8-LNK1-COR27,28 protein complex by time-of-day affinity purification-mass spectrometry, and found that LNK1 and LNK2 expression respond to temperature entrainment (Sorkin et al. 2023).
Circadian clock components provide the genetic basis for crops adaptation to agricultural environments.For example, LNK2 was found to co-localize with the circadian period length QTL in cultivated tomato (Muller et al. 2018).The frequency of LNK2 deletion increased during tomato domestication, which slowed down the clock pace, i.e., partial mutation of LNK2 lengthened the period length of endogenous circadian rhythms.Muller et al. also revealed the evolutionary trajectory of EMP-FINDLICHER IM DUNNKELROTEN LICHT 1 (EID1) and LNK2 mutations during domestication and breeding with the analysis of a subset of 274 tomato accessions.EID1 encodes an F-box protein that acts in the PHYA-dependent light signaling pathway in Arabidopsis.Soybean GmEID1 interacts with J (a homolog of ELF3), which regulates photoperiodic flowering and yield in a variety of crops, including soybean, barley, and rice (Faure et al. 2012;Andrade et al. 2022;Qin et al. 2023).In summary, the mechanism of circadian oscillator protein complex in the response to environmental timing cues (inputs) and clock-gating physiological processes (outputs) deserves further investigation.

Fig. 1
Fig.1Circadian oscillators respond to the fluctuations in ambient temperature.Circadian transcription factors RVE4, RVE8 and coactivators LNK3, LNK4 activate DREB1s, COR15A, and COR47 expression to achieve the chilling and freezing tolerance.Cold-induced phosphorylation of LNK3,4 proteins contribute to the low temperature response of the RVE4,8-LNK3,4 complex.In response to heat shock and thermal stress, RVE4 and RVE8 recruit LNK1 and LNK2 and activate ERFs expression to obtain thermotolerance.In addition, the LNK1,2-interacting protein CCA1 directly regulate the transcription of COR27 and COR28