
==== Front
Plant Commun
Plant Commun
Plant Communications
2590-3462
Elsevier

S2590-3462(24)00289-X
10.1016/j.xplc.2024.100981
100981
Research Article
The PRR–EC complex and SWR1 chromatin remodeling complex function cooperatively to repress nighttime hypocotyl elongation by modulating PIF4 expression in Arabidopsis
Won Jin Hoon 15
Park Jeonghyang 25
Lee Hong Gil 13
Shim Sangrae 4
Lee Hongwoo 1
Oh Eunkyoo ekoh@korea.ac.kr
2∗
Seo Pil Joon pjseo1@snu.ac.kr
13∗∗
1 Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea
2 Department of Life Sciences, Korea University, Seoul 02841, Republic of Korea
3 Plant Genomics and Breeding Institute, Seoul National University, Seoul 08826, Republic of Korea
4 Department of Forest Resources, Kangwon National University, Chuncheon 24341, Republic of Korea
∗ Corresponding author ekoh@korea.ac.kr
∗∗ Corresponding author pjseo1@snu.ac.kr
5 These authors contributed equally to this article.

29 5 2024
09 9 2024
29 5 2024
5 9 10098119 1 2024
26 2 2024
27 5 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The circadian clock entrained by environmental light–dark cycles enables plants to fine-tune diurnal growth and developmental responses. Here, we show that physical interactions among evening clock components, including PSEUDO-RESPONSE REGULATOR 5 (PRR5), TIMING OF CAB EXPRESSION 1 (TOC1), and the Evening Complex (EC) component EARLY FLOWERING 3 (ELF3), define a diurnal repressive chromatin structure specifically at the PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) locus in Arabidopsis. These three clock components act interdependently as well as independently to repress nighttime hypocotyl elongation, as hypocotyl elongation rate dramatically increased specifically at nighttime in the prr5-1 toc1-21 elf3-1 mutant, concomitantly with a substantial increase in PIF4 expression. Transcriptional repression of PIF4 by ELF3, PRR5, and TOC1 is mediated by the SWI2/SNF2-RELATED (SWR1) chromatin remodeling complex, which incorporates histone H2A.Z at the PIF4 locus, facilitating robust epigenetic suppression of PIF4 during the evening. Overall, these findings demonstrate that the PRR–EC–SWR1 complex represses hypocotyl elongation at night through a distinctive chromatin domain covering PIF4 chromatin.

This study reports that the core circadian clock components PRR5, TOC1, and ELF3 interact with each other and cooperatively regulate PIF4 expression, and the PRR–EC complex diurnally recruits the SWR1 complex to PIF4 chromatin to repress its expression in the evening.

Key words

hypocotyl elongation
circadian clock
PRR
Evening Complex
histone H2A.Z
SWR1 complex
Published: May 29, 2024
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pmcIntroduction

Plants have evolved to maintain an internal rhythm with a period of approximately 24 h. Light–dark and hot–cold temperature cycles are the major environmental cues that entrain the circadian clock, which ensures time-of-day-specific responses, including growth, stress responses, and metabolism (Covington and Harmer 2007; Seo and Mas 2015; Bonnot and Nagel 2021; Steed et al., 2021), in synchrony with diurnal environmental fluctuations. The representative study model for circadian regulation of plant growth is diurnal elongation. The rhythmic hypocotyl elongation of Arabidopsis (Arabidopsis thaliana) is intricately determined by the diurnally regulated PHYTOCHROME-INTERACTING FACTOR (PIF) basic helix-loop-helix (bHLH) transcription factors, which promote cell elongation through activation of both auxin and brassinosteroid biosynthesis and signaling-related genes (Gray William et al., 1998; Nozue et al., 2007; Oh et al., 2012; Sun et al., 2012; Martínez et al., 2018).

PIFs are regulated at multiple levels, reaching their maximum transcriptional activity at a specific time of day depending on the photoperiod conditions (Nozue et al., 2007; Yamashino et al., 2013; Chung et al., 2020). The plant red/far-red photoreceptor phytochrome B (phyB) is converted from the inactive red-light-absorbing Pr form into the active far-red-light-absorbing Pfr form upon light exposure during the day, resulting in its translocation into the nucleus (Klose et al., 2015), where it directly interacts with PIFs (Huq and Quail 2002). Interactions between active phyB (Pfr) and PIFs facilitate phosphorylation of PIFs, leading to their degradation and inactivation (Huq and Quail 2002; Lorrain et al., 2008; Park et al., 2018). By contrast, under dark conditions, phyB is sequestered in the cytoplasm after reversion to the inactive Pr form, which results in the gradual accumulation of PIFs during the night (Huq and Quail 2002; Nozue et al., 2007; Lorrain et al., 2008).

Most core clock components either bind to PIF loci or interact with PIFs to restrict PIF activities specifically at dawn (Zhu et al., 2016; Martín et al., 2018; Nohales et al., 2019). Five members of the Arabidopsis PSEUDO-RESPONSE REGULATOR (PRR) family, PRR9, PRR7, PRR5, PRR3, and PRR1 (also named TIMING OF CAB2 EXPRESSION 1 [TOC1]), are integral components of the circadian oscillator, and they share a considerable number of target genes, including PIFs (Soy et al., 2016; Zhu et al., 2016; Martín et al., 2018). PRR9 and PRR7 directly bind to the PIF4 and PIF5 promoters and repress transcription during the daytime (Nakamichi et al., 2012; Liu et al., 2013), whereas PRR5 and TOC1 repress PIF4 and PIF5 expression during the evening, allowing these PIFs to be temporally upregulated at the end of the night (Nozue et al., 2007; Niwa et al., 2009; Nakamichi et al., 2012; Liu et al., 2013; Li et al., 2020). TOC1 also physically interacts with PIF3 and PIF4, inhibiting PIF activities and thus regulating growth-promoting genes, including CYCLING DOF FACTOR 5 (CDF5) (Soy et al., 2016; Zhu et al., 2016; Martín et al., 2018). In addition, the Evening Complex (EC)—composed of EARLY FLOWERING 3 (ELF3), ELF4, and LUX ARRHYTHMO (LUX), which peak at dusk (Hicks et al., 2001; Liu et al., 2001; Doyle et al., 2002; Hazen et al., 2005; Onai and Ishiura, 2005; Nusinow et al., 2011)—also integrates circadian signals into PIF4 and PIF5 expression for diurnal growth regulation (Nusinow et al., 2011; Lu et al., 2012; Nomoto et al., 2012; Box et al., 2015; Murcia et al., 2022). LUX recruits ELF3 and ELF4 at the PIF4 and PIF5 loci (Nusinow et al., 2011), ensuring the transcriptional repression of these PIFs in the evening (Silva et al., 2020). Moreover, protein–protein interactions of ELF3 with PIFs also interfere with the target DNA-binding activity of PIFs in an EC-independent manner (Nieto et al., 2015). Overall, PIF activities are highest at dawn, resulting from the coordinated actions of various circadian clock components and photoreceptors.

EC function involves various epigenetic mechanisms that modulate circadian-regulated gene expression. For instance, the EC interacts with multiple chromatin modifiers such as HISTONE DEACETYLASE 9 (HDA9) and Jumonji C domain-containing histone demethylase JUMONJI 29 (JMJ29) (Lee et al., 2019; Park et al., 2019; Lee and Seo 2021). In addition, the EC also works with the SWI2/SNF2-RELATED 1 (SWR1) chromatin remodeling complex, which consists of PHOTOPERIOD INDEPENDENT EARLY FLOWERING 1 (PIE1), ACTIN-RELATED PROTEIN 6 (ARP6), and SERRATED LEAVES AND EARLY FLOWERING (SEF) (March-Díaz and Reyes 2009; Aslam et al., 2019; Lei and Berger 2020), to diurnally exchange canonical histone H2A with the histone variant H2A.Z at target loci. Because the enrichment of H2A.Z at gene body regions frequently leads to transcriptional silencing (Coleman-Derr and Zilberman 2012; Kumar 2018; Lei and Berger 2020), the global H2A.Z deposition that occurs at EC target genes around dusk mediates circadian-dependent control of gene expression (Tong et al., 2020).

Although circadian clock components are extensively linked to each other in Arabidopsis (Li et al., 2020), it has remained unclear how these proteins work together to control diurnal growth. Here, we demonstrate that the core circadian clock components PRR5, TOC1, and ELF3 interact with each other and interdependently regulate PIF4 expression. In addition, we show that the PRR–EC complex diurnally recruits the SWR1 complex to PIF4 chromatin to repress its expression around evening time, thus enabling plants to adjust their growth according to photoperiod. Overall, our study reveals that the core circadian complex facilitates circadian growth responses via repressive nuclear chromatin domains.

Results

ELF3, PRR5, and TOC1 exhibit partial interdependence in suppression of hypocotyl elongation at night

PRR5, TOC1, and ELF3 are known to regulate hypocotyl elongation by controlling PIF expression (Sato et al., 2002; Kaczorowski and Quail, 2003; Yamamoto et al., 2003; Nusinow et al., 2011; Huang et al., 2012; Nakamichi et al., 2012; Nieto et al., 2015; Soy et al., 2016; Zhu et al., 2016; Martín et al., 2018). In addition, the expression levels of PRR5, TOC1, and genes encoding EC components temporally coincide over a diurnal cycle (Más et al., 2003; Nakamichi et al., 2010; Nusinow et al., 2011) and reach a peak during the evening (Supplemental Figure 1), suggesting that the proteins encoded by these genes may work together in the control of hypocotyl elongation. To examine the association of their functions in hypocotyl elongation, we measured the hypocotyl lengths of seedlings of prr5-1, toc1-21, and elf3-1 single mutants and higher-order mutants. The mean hypocotyl lengths of toc1-21 and elf3-1 mutants were twice and four times that of wild-type Col-0, respectively, under neutral-day (ND) conditions (12-h light/12-h dark) (Figure 1A and 1B), whereas the prr5-1 mutant displayed a marginal difference in hypocotyl length compared with the wild type (Figure 1A and 1B). Notably, hypocotyl length was significantly greater in the prr5-1 toc1-21, prr5-1 elf3-1, toc1-21 elf3-1, and prr5-1 toc1-21 elf3-1 double and triple mutants than in their corresponding single mutants (Figure 1A and 1B) (Li et al., 2020). The hypocotyl lengths of higher-order mutants were mostly close to that of the elf3-1 mutant (Figure 1A and 1B), suggesting that ELF3 has the most substantial effect on hypocotyl elongation and that the functions of PRR5 and TOC1 may be partially dependent on ELF3.Figure 1 PRR5, TOC1, and ELF3 suppress hypocotyl growth at night.

(A) Representative image of 7-day-old Col-0, prr5-1, toc1-21, prr5-1 toc1-21, elf3-1, prr5-1 elf3-1, toc1-21 elf3-1, and prr5-1 toc1-21 elf3-1 seedlings grown at 22°C under neutral-day (ND) conditions. Scale bar, 5 mm.

(B) Hypocotyl lengths of the genotypes shown in (A). Seedlings were grown for 7 days at 22°C under ND conditions. Hypocotyl lengths (n > 30 for each genotype) were measured using ImageJ. Numbers indicate the ratio of hypocotyl length between mutants and the wild type (Col-0/mutants).

(C) Hypocotyl elongation rate during the day and night. Seedlings (n > 10 for each genotype) were grown at 22°C under ND conditions. All time-lapse images were analyzed with ImageJ. Yellow and gray bars indicate average hypocotyl elongation rate during the day and night, respectively. In (B) and (C), values are means ± SD; different lowercase letters indicate significant differences based on one-way ANOVA and Tukey’s test (p < 0.05).

Given the overlapping temporal expression patterns of PRR5, TOC1, and ELF3, which peaked in the evening (Supplemental Figure 1), we suspected that the increased hypocotyl elongation seen in the higher-order mutants might be caused by growth derepression during the dark period. We thus measured hypocotyl elongation rates using time-lapse imaging, which revealed that hypocotyl elongation of prr5-1 toc1-21, prr5-1 elf3-1, toc1-21 elf3-1, and prr5-1 toc1-21 elf3-1 seedlings is promoted specifically during the nighttime (Figure 1C, and Supplemental Figure 2). By contrast, the hypocotyl elongation rate of all mutants examined was indistinguishable from that of the wild type during the daytime (Figure 1C; and Supplemental Figure 2). Nighttime hypocotyl elongation regulated by PRR5 and TOC1 seems to be partly dependent on ELF3, as the nighttime hypocotyl elongation rates of higher-order mutants were only slightly higher than that of elf3-1 (Figure 1C and Supplemental Figure 2). Overall, these results suggest a partial functional interdependence between PRR5, TOC1, and ELF3 in repression of hypocotyl elongation at night.

PRR5, TOC1, and ELF3 co-regulate PIF4 expression at night

To investigate how PRR5, TOC1, and ELF3 repress hypocotyl elongation at night, we obtained published datasets from chromatin immunoprecipitation sequencing (ChIP-seq) experiments performed with transgenic plants that accumulated epitope-tagged PRR5, TOC1, or ELF3 (Huang et al., 2012; Nakamichi et al., 2012; Tong et al., 2020). We then identified genes co-targeted by the three proteins (Figure 2A; Supplemental Table 1). Among the seven candidate genes whose loci are bound by PRR5, TOC1, and ELF3, we focused on PIF4 (Figure 2A and Supplemental Table 1), which functions as a hub integrating light and temperature signals into plant growth programs (Koini et al., 2009; Sun et al., 2012; Zhao and Bao, 2021). We observed that the two PRRs and ELF3 bind to the same region in the PIF4 promoter (Figure 2B). The overlapping binding sites contain both a G-box motif (CACGTG, −660 bp) (Gendron et al., 2012) and a LUX binding site (LBS, GATTCG, −376 bp) (Helfer et al., 2011), which are known cis elements bound by PRRs and the EC, respectively. Our ChIP quantitative PCR (ChIP–qPCR) analysis using proPRR5:PRR5-eGFP (encoding PRR5 fused to the enhanced green fluorescent protein), TMG-YFP (a TOC1 minigene cloned in frame and upstream of the yellow fluorescent protein), and elf3-1 proELF3:ELF3-MYC transgenic plants confirmed that the three proteins target similar regions in the PIF4 promoter, specifically at dusk (Figure 2C–2E).Figure 2 PRR5, TOC1, and ELF3 bind to the PIF4 locus and synergistically repress its expression at night.

(A) Venn diagram of overlapping and distinct genes targeted by PRR5, TOC1, and ELF3. Target genes were defined as genes with ChIP-seq peaks up to 1 kb upstream of the transcription start site and up to 1 kb downstream of the translation end site.(B) Integrated genome browser view of ChIP-seq peaks at the PIF4 locus (At2g43010).(C) Genomic structure of the PIF4 locus. Blue boxes indicate 5′ and 3′ untranslated regions. Yellow boxes indicate exons. Arrows indicate the regions analyzed by qPCR following chromatin immunoprecipitation (ChIP). Black and gray vertical lines indicate the G-box and the LUX binding site (LBS), respectively.(D and E) Binding of PRR5, TOC1 (D), and ELF3 (E) at the PIF4 locus. Two-week-old seedlings entrained under ND cycles were transferred to continuous-light conditions and harvested at zeitgeber time (ZT) 4 and ZT16 for ChIP–qPCR analysis. The enrichment of DNA was normalized to that at the eIF4a locus. Values are means ± SEM (∗p < 0.05; Student’s t-test).(F) PIF4 expression in Col-0, prr5-1, toc1-21, prr5-1 toc1-21, elf3-1, prr5-1 elf3-1, toc1-21 elf3-1, and prr5-1 toc1-21 elf3-1 seedlings. Two-week-old seedlings grown under ND conditions were harvested at ZT16. Transcript levels were quantified using RT–qPCR. Gene expression values were normalized against eIF4a expression. Fold changes (FCs) in expression were calculated relative to the value of Col-0 seedlings and are presented as log2FC values. Values are means ± SEM from biological triplicates. Different lowercase letters indicate significant differences based on one-way ANOVA and Tukey’s test (p < 0.05).(G) Transient expression analysis using Arabidopsis mesophyll protoplasts. Effector constructs for PRR5, TOC1, and ELF3 expression and reporter constructs containing the promoter and genomic sequence of the PIF4 gene were transiently co-expressed in wild-type protoplasts. GUS activity was normalized to LUC activity. Values are means ± SEM from biological triplicates. Numbers above bars indicate FCs relative to the empty-vector control.

We determined the diurnal expression pattern of PIF4 in prr5-1, toc1-21, and elf3-1 single mutants and prr5-1 toc1-21, prr5-1 elf3-1, toc1-21 elf3-1, and prr5-1 toc1-21 elf3-1 higher-order mutants by reverse-transcription qPCR (RT–qPCR). PIF4 transcript levels showed negligible differences in prr5-1, toc1-21, and elf3-1 single mutants compared with the wild type during the daytime (at zeitgeber time [ZT] 4 and ZT8) (Supplemental Figure 3). By contrast, PIF4 expression at ZT16 was higher in all single and higher-order mutants than in the wild type during the nighttime (Figure 2F and Supplemental Figure 3) in proportion to the extent of their hypocotyl elongation and their nighttime elongation rate (Figure 1A–1C, and Supplemental Figure 2). Notably, PIF4 expression in higher-order mutants was equivalent to that in the elf3-1 mutant (Figure 2F).

To further support the overlapping functions of PRRs and ELF3 in regulation of PIF4 expression, we generated the proPIF4:gPIF4-GUS reporter construct by fusing the genomic sequence of PIF4, including ∼2 kb of upstream promoter and genic regions, in frame and upstream of the β-glucuronidase (GUS) sequence. We then co-transfected Arabidopsis protoplasts with this reporter construct and effector constructs overexpressing PRR5 (35S:HA-PRR5), TOC1 (35S:HA-TOC1), and/or ELF3 (35S:MYC-ELF3). We determined that PIF4 expression is repressed in protoplasts overexpressing PRR5, TOC1, or ELF3 (Figure 2G). In addition, simultaneous overexpression of PRR and ELF3 further repressed PIF4 expression (Figure 2G). It is notable that, because ELF3 acts as a component of the EC (Nusinow et al., 2011), the repression of PIF4 by PRR5, TOC1, and/or ELF3 was most likely dependent on LUX (Supplemental Figure 4), which determines binding of the EC to target DNA sites (Nusinow et al., 2011). Overall, these results indicate that PRRs and ELF3 bind to the PIF4 promoter and coordinately repress its transcription during the night.

PRR5 and TOC1 interact with ELF3

Given the overlapping binding sites of PRR5, TOC1, and ELF3 at the PIF4 locus and their cooperative functions in repression of hypocotyl elongation, we hypothesized that these three circadian components may form a protein complex. Indeed, yeast two-hybrid (Y2H) assays showed that PRR5 physically interacts with ELF3 and TOC1 (Figure 3A). To further confirm the interactions between PRRs and the EC in vivo, we conducted bimolecular fluorescence complementation (BiFC) assays using Arabidopsis protoplasts. Accordingly, we cloned the coding sequences of PRR5 and TOC1 in frame with a sequence encoding the N-terminal half of the yellow fluorescent protein (nYFP), and we cloned the coding sequence of ELF3 in frame with a sequence encoding the C-terminal half of YFP (cYFP). We observed strong YFP signals in the nuclei of Arabidopsis protoplasts co-transfected with the combinations PRR5-nYFP+cYFP-ELF3 and TOC1-nYFP+cYFP-ELF3, but we detected no fluorescence signal in protoplasts transfected with the negative controls (Figure 3B).Figure 3 PRRs physically interact with ELF3.

(A) Yeast two-hybrid (Y2H) assays. Y2H assays were performed with PRR5, TOC1, and ELF3 fused to either the DNA-binding domain (BD) or the activation domain (AD) of GAL4. Full-length GAL4 was used as a positive control. Transformed yeast cells were grown on selective medium. –LW indicates synthetic defined (SD) medium lacking Leu and Trp. –LWHA indicates SD medium lacking Leu, Trp, His, and Ade.(B) Bimolecular fluorescence complementation (BiFC) assays using Arabidopsis protoplasts. N-terminal or C-terminal fragments of YFP (nYFP or cYFP, respectively) were fused to PRR5, TOC1, or ELF3. Appropriate pairs of constructs were co-transfected along with a nuclear marker (NLS-mCherry, nucleus). Scale bars, 20 μm.(C) CoIP assays. 35S:HA-PRR5, 35S:HA-TOC1, and 35S:MYC-ELF3 constructs were transfected into Arabidopsis mesophyll protoplasts. Epitope-tagged proteins were immunoprecipitated with anti-HA magnetic beads and detected by immunoblotting with anti-HA and anti-MYC antibodies.

We also performed co-immunoprecipitation (coIP) assays using Arabidopsis protoplasts transfected with HA-PRR5 or HA-TOC1, alone or together with MYC-ELF3. When using an anti-MYC antibody for immunoprecipitation, we detected both HA-PRR5 and HA-TOC1 among the immunoprecipitates (Figure 3C), indicating in vivo interactions between PRRs and the EC.

PRRs and the EC act interdependently in the control of hypocotyl elongation

To further examine whether PRRs and ELF3 interdependently regulate PIF4 expression and hypocotyl elongation, we crossed transgenic plants overexpressing PRR5 or TOC1 with the elf3-1 mutant. Overexpression of PRR5 resulted in shorter hypocotyls with significantly lower PIF4 expression (at ZT16) in the wild-type background (Figure 4A–4D and Supplemental Figure 5). Notably, the effect of PRR5 overexpression on hypocotyl elongation completely disappeared in the elf3-1 mutant background (elf3-1 PRR5-ox) (Figure 4A–4D, and Supplemental Figure 5). This was also the case in the lux-6 mutant background (lux-6 PRR5-ox) (Supplemental Figure 6). Similarly, the function of TOC1 in repressing hypocotyl elongation was also largely dependent on ELF3. Indeed, whereas TOC1-ox seedlings had a shorter hypocotyl than wild-type seedlings, elf3-1 TOC1-ox seedlings had a hypocotyl length intermediate between those of TOC1-ox and elf3-1 (Figure 4A–4D and Supplemental Figure 5). These results indicate that PRR functions are dependent on the EC in the control of PIF4-mediated hypocotyl elongation. However, because the lux-6 PRR5-ox and elf3-1 TOC1-ox mutants had shorter hypocotyls than the lux-6 and elf3-1 mutants (Figure 4A–4D and Supplemental Figure 6), it appears that the PRRs may also repress hypocotyl elongation independently of the EC.Figure 4 PRR-dependent repression of hypocotyl elongation occurs in an ELF3-dependent manner.

(A) Representative image of 7-day-old Col-0, elf3-1, PRR5-ox, elf3-1 PRR5-ox, TOC1-ox, and elf3-1 TOC1-ox seedlings grown at 22°C under ND conditions. Scale bar, 5 mm.(B) Mean hypocotyl lengths of the seedlings shown in (A). Seedlings were grown for 7 days under ND conditions. Hypocotyl lengths (n > 30 in each genotype) were measured using ImageJ. Numbers indicate the ratio of hypocotyl length between genotypes.(C) Hypocotyl elongation rate during the day and night. Seedlings (n > 10 in each genotype) were grown at 22°C under ND conditions. All time-lapse images were analyzed using ImageJ. Yellow and gray bars indicate average hypocotyl elongation rate during the day and night, respectively.(D) PIF4 expression in Col-0, elf3-1, PRR5-ox, elf3-1 PRR5-ox, TOC1-ox, and elf3-1 TOC1-ox seedlings. Two-week-old seedlings grown under ND conditions were harvested at ZT4 and ZT16. Transcript levels were analyzed by RT–qPCR. Gene expression values were normalized against eIF4a expression. Relative gene expression values are represented as FC (upper panel, ZT4) or log2FC (lower panel, ZT16) relative to the wild type (Col-0). In (B–D), values are means ± SD from biological triplicates. Statistical significance was determined by Student’s t-test (∗p < 0.05; NS, not significant).(E and F) Representative images (left) and mean hypocotyl lengths (right) of seedlings grown under continuous red light for 5 days. Different lowercase letters indicate significant differences based on one-way ANOVA and Tukey’s test (p < 0.05). Values are means ± SD; n = 15 (E) and n = 12 (F) in each genotype. Numbers indicate the ratio of hypocotyl length between genotypes. prr-q, prr9 prr7 prr5 toc1 quadruple mutant.

(G) Transient expression analysis using Arabidopsis mesophyll protoplasts. The effector and reporter constructs were co-transfected into protoplasts isolated from wild-type, elf3-1, or prr5-1 toc1-21 seedlings. GUS activity was normalized to LUC activity. Values are means ± SEM from biological triplicates. Numbers indicate the FC relative to the empty-vector control.

Because hypocotyl elongation under ND conditions was reduced only slightly by overexpression of PRR5 or TOC1, we also grew the seedlings under continuous-red-light conditions, in which hypocotyl elongation is more sensitive to levels of PRR5 and TOC1 (Sato et al., 2002). The effect of PRR5 or TOC1 overexpression on the repression of hypocotyl elongation was more pronounced under red-light conditions than under ND conditions (Figure 4E). The PRR-mediated inhibition of hypocotyl elongation was largely suppressed in the elf3-1 and lux-6 mutant backgrounds (Figure 4E and Supplemental Figure 7). Consistent with this result, expression of PIF4 and PIF4 target genes was repressed in PRR5-ox and TOC1-ox but derepressed in the elf3-1, elf3-1 PRR5-ox, and elf3-1 TOC1-ox backgrounds (Supplemental Figure 8).

To understand whether ELF3 also requires PRRs for PIF4 repression, we generated transgenic plants overexpressing ELF3 (ELF3-ox) in both the wild-type and a prr quadruple (prr9 prr7 prr5 toc1; prr-q) mutant background and analyzed their hypocotyl elongation phenotypes. Under continuous red-light conditions, ELF3-ox seedlings displayed shorter hypocotyls with lower expression of PIF4 and PIF4 target genes (Figure 4F and Supplemental Figure 9); however, the repressive action of ELF3 was impaired in the prr-q mutant background (Figure 4F and Supplemental Figure 9), as shown by the comparably long hypocotyls of prr-q and prr-q ELF3-ox seedlings.

To confirm the interdependence of PRRs and ELF3 in repression of PIF4 expression, we transfected effector constructs overexpressing PRR5, TOC1, or ELF3, along with the proPIF4:gPIF4-GUS reporter construct, into mesophyll protoplasts isolated from wild-type, elf3-1, or prr5-1 toc1-21 seedlings (Figure 4G). We then measured relative GUS activity in these cells and found that PIF4 transcription is repressed by overexpression of one or more PRRs in the wild-type background; notably, this repression of PIF4 transcription by PRRs is lost in the elf3-1 mutant background (Figure 4G). Similarly, we observed that the repression of PIF4 transcription by ELF3 is compromised in the prr5-1 toc1-21 mutant background (Figure 4G). Overall, these results indicate that PRRs and the EC interdependently repress PIF4 transcription and, thus, hypocotyl elongation.

Diurnal deposition of histone H2A.Z at the PIF4 locus is mediated by the PRR–EC complex

The EC has been shown to interact with the SWR1 complex to establish repressive chromatin domains by catalyzing histone H2A.Z exchange, specifically at night (Tong et al., 2020). We therefore investigated whether the PRR–EC complex also requires SWR1 to shape diurnal PIF4 expression. To this end, we analyzed the distribution of histone H2A.Z over the chromatin of genes targeted by PRRs and/or ELF3 (Supplemental Table 1). We observed an increase in H2A.Z signals at ELF3 target genes at ZT16 compared with ZT4 (Supplemental Figure 10); moreover, the nighttime-specific increase in H2A.Z signals was compromised in the elf3-1 mutant (Supplemental Figure 10), as demonstrated previously (Tong et al., 2020). In addition, although H2A.Z deposition over the chromatin of genes targeted solely by either PRR5 or TOC1 was comparable to that of randomly selected control genes, regardless of time, the chromatin of genes bound by both PRR5 and TOC1 showed a substantial increase in H2A.Z signal compared with that of control genes, specifically at ZT16 (Supplemental Figure 10), suggesting that PRRs, like ELF3, contribute to diurnal H2A.Z deposition. Notably, we also analyzed genes co-targeted by ELF3 and PRR5 or TOC1 and established that H2A.Z levels over the chromatin of these co-targeted genes are further elevated compared with those targeted by PRR5, TOC1, or ELF3 alone (Supplemental Figure 10). Moreover, we detected the highest H2A.Z enrichment over the chromatin of genes bound by PRR5, TOC1, and ELF3, and this synergistic H2A.Z enrichment was compromised in the elf3-1 mutant (Supplemental Figure 10), indicating that PRR5, TOC1, and EC coordinately modulate the deposition of H2A.Z at co-targeted loci.

To further corroborate the synergistic functions of PRRs and the EC in diurnal H2A.Z deposition at the PIF4 locus, we examined H2A.Z enrichment in elf3-1, prr5-1 toc1-21, and prr5-1 toc1-21 elf3-1 mutants. In the wild type, H2A.Z enrichment at the PIF4 locus was substantially higher at night than during the day (Supplemental Figure 11). The night-specific deposition of H2A.Z at the PIF4 locus was lower in the elf3-1 and prr5-1 toc1-21 mutants than in the wild type (Figure 5A–5C and Supplemental Figure 12). Moreover, we observed that H2A.Z deposition at the PIF4 locus is synergistically regulated by PRR5, TOC1, and the EC, as the decrease in H2A.Z deposition covered a broader region of the PIF4 locus in the prr5-1 toc1-21 elf3-1 mutant than in the elf3-1 and prr5-1 toc1-21 mutants (Figure 5A–5D and Supplemental Figure 12).Figure 5 Histone H2A.Z deposition is compromised in mutants of circadian clock components.

(A) Genomic structure of the PIF4 locus. Arrows indicate regions analyzed by ChIP–qPCR. Black and gray vertical lines indicate the G-box and the LBS, respectively.(B–D) H2A.Z levels at the PIF4 locus in elf3-1(B), prr5-1 toc1-21(C), and prr5-1 toc1-21 elf3-1(D) mutant seedlings. Two-week-old seedlings entrained under ND cycles were harvested at ZT4 and ZT16. Enrichment of precipitated DNA was analyzed by ChIP–qPCR and normalized to that of the eIF4a locus. Values are means ± SEM from biological triplicates. Different lowercase letters indicate significant differences based on one-way ANOVA and Tukey’s test (p < 0.05).

We speculated that the synergistic functions of PRR5, TOC1, and the EC in H2A.Z deposition are likely related to their interactions with the SWR1 complex. Y2H assays showed that ELF3 interacts with the SWR1 complex component SEF as well as the SANT domain of PIE1 (Figure 6A). Likewise, PRR5 and TOC1 interact with the SANT domain of PIE1 (Figure 6A). We also performed coIP experiments using Arabidopsis protoplasts transfected with the constructs 35S:FLAG-PIE1, 35S:HA-PRR5, 35S:HA-TOC1, and/or 35S:MYC-ELF3. Following immunoprecipitation with an anti-FLAG antibody, we detected PRR5, TOC1, and ELF3 among the immunoprecipitates, indicating that PRR5, TOC1, and ELF3 interact with the SWR1 complex in vivo (Figure 6B, and Supplemental Figure 13). To quantify the strength of this interaction, we performed split-luciferase assays using Arabidopsis protoplasts. We determined that the ELF3–PIE1 interaction is strengthened by co-expression of PRR5 or TOC1 (Figure 6C). Moreover, simultaneous expression of all three constructs (35S:MYC-ELF3, 35S:HA-PRR5, 35S:HA-TOC1) further enhanced the interaction of their encoded proteins with the SWR1 complex (Figure 6C), indicating that the formation of a PRR–EC–SWR1 complex is responsible for robust H2A.Z deposition.Figure 6 The PRR–EC complex interacts with the SWR1 complex.

(A) Interaction between PIE1 and ELF3 or PRRs. Y2H assays were performed with PRR5, TOC1, and ELF3 fused to the GAL4 AD; the SWR1 complex components SEF, ARP6, PIE1SANT, and PIE1SNF2 were fused to the GAL4 DNA BD. Full-length GAL4 was used as a positive control. –LW indicates SD medium lacking Leu and Trp; –LWHA indicates SD medium lacking Leu, Trp, His, and Ade.(B) CoIP assays. 35S:FLAG-PIE1, 35S:HA-PRR5, 35S:HA-TOC1, and/or 35S:MYC-ELF3 constructs were transfected into Arabidopsis protoplasts. Epitope-tagged proteins were immunoprecipitated with anti-FLAG magnetic beads and detected by immunoblotting with anti-FLAG, anti-HA, or anti-MYC antibodies.(C) Split-luciferase assays. The N-terminal fragment of luciferase was fused to PRR5, TOC1, or ELF3, and the C-terminal fragment of luciferase was fused to PIE1. The PRR5, TOC1, or ELF3 effector plasmids, the LUC reporter plasmid, and GUS-expressing internal control plasmids were co-transfected into Arabidopsis protoplasts. LUC activity was normalized to GUS activity. Values are means ± SEM from biological triplicates. Different lowercase letters indicate significant differences based on one-way ANOVA and Tukey’s test (p < 0.05).

Repression of hypocotyl elongation by the PRR–EC complex depends on the SWR1 complex

Consistent with the link between SWR1 and the PRRs and the EC, the SWR1 complex plays a role in hypocotyl elongation (Kumar and Wigge 2010; Tong et al., 2020; Mao et al., 2021; Wei et al., 2021; Xue et al., 2021). In agreement with this notion, the hypocotyls of sef-1 and arp6-1 mutant seedlings were longer than those of wild-type seedlings when grown under ND conditions (Figure 7A and 7B). Furthermore, PIF4 expression was significantly higher in the arp6-1 mutant than in the wild type during the nighttime (Figure 7C), corresponding to a strong decline in H2A.Z enrichment across the PIF4 locus (Figure 7D).Figure 7 The SWR1 complex represses hypocotyl elongation through PIF4 regulation.

(A) Representative image of 7-day-old Col-0, sef-1, and arp6-1 seedlings grown under ND conditions. Scale bar, 5 mm.(B) Hypocotyl lengths of the seedlings shown in (A). Hypocotyl lengths (n > 30 in each genotype) were measured using ImageJ. Values are means ± SD. Different lowercase letters indicate significant differences based on one-way ANOVA and Tukey’s test (p < 0.05).(C) Diurnal PIF4 expression in arp6-1. Seven-day-old seedlings grown under ND conditions were harvested at 4-h intervals from ZT0 to ZT20. Transcript levels were analyzed by RT–qPCR and are represented as FC relative to the value of the wild type. Biological triplicates were averaged. Error bars indicate the SD of the mean. Different lowercase letters indicate significant differences based on one-way ANOVA and Tukey’s test (p < 0.05).(D) Histone H2A.Z distribution at the PIF4 locus. H2A.Z signals in Col-0, arp6 (GSE108450), and pie1 (GSE139459) seedlings were visualized using the genome browser provided by iRegNet (Shim et al., 2021).(E) Genomic structure of the PIF4 locus. Arrows indicate regions analyzed by ChIP–qPCR. Black and gray vertical lines indicate the G-box and the LBS, respectively.(F) Histone H2A.Z levels at the PIF4 locus in Col-0, PRR5-ox, arp6-1, and arp6-1 PRR5-ox. Two-week-old seedlings entrained under ND cycles were harvested at ZT16. The enrichment of precipitated DNA was analyzed by ChIP–qPCR and normalized to that of the eIF4a locus. Values are means ± SEM from biological triplicates.(G) PIF4 expression in Col-0, PRR5-ox, arp6-1, and arp6-1 PRR5-ox seedlings. Seven-day-old seedlings grown under ND conditions were harvested at ZT16. Transcript levels were analyzed by RT–qPCR. Values are means ± SD from biological triplicates. The numbers indicate the ratio of PIF4 transcript levels between the indicated genotypes (Col-0/PRR5-ox and arp6-1/arp6-1 PRR5-ox).(H and I) Hypocotyl lengths of seedlings grown under ND conditions for 7 days (H) or in continuous red light for 5 days (I). Values are means ± SD (n > 20 in each genotype). Numbers indicate the ratio of hypocotyl length between the indicated genotypes (Col-0/PRR5-ox and arp6-1/arp6-1 PRR5-ox). In (F–I), different lowercase letters indicate significant differences based on one-way ANOVA and Tukey’s test (p < 0.05).(J) Working diagram showing diurnal regulation of PIF4 expression by the PRR–EC–SWR1 complex. The circadian clock components PRR5, TOC1, and ELF3 form a protein complex and interact with the SWR1 complex (SWR1-C) to repress PIF4 expression, thereby inhibiting hypocotyl elongation at night.

We therefore examined whether the control of PIF4 repression and hypocotyl elongation mediated by the PRR–EC complex was dependent on the SWR1 complex. Because functional interdependency between the EC and SWR1 has been demonstrated previously (Tong et al., 2020), we focused on the genetic relationship between PRR5 and SWR1 in this study. We performed ChIP–qPCR analysis with an anti-H2A.Z antibody on PRR5-ox, arp6-1, and arp6-1 PRR5-ox seedlings. This revealed that overexpression of PRR5 significantly increases H2A.Z deposition at the PIF4 locus, but this effect on H2A.Z deposition is diminished in the arp6-1 mutant (Figure 7E and 7F). In agreement with these results, the repression of PIF4 expression and hypocotyl elongation caused by PRR5 overexpression was significantly attenuated in the arp6-1 mutant background (Figure 7G–7I). We concluded that the PRR–EC complex represses PIF4 transcription in concert with the SWR1 complex, repressing hypocotyl elongation at night (Figure 7J).

Discussion

The circadian clock is composed of multiple transcriptional negative feedback loops established by a multitude of transcription factors (Harmer and Kay, 2005; Helfer et al., 2011; Nusinow et al., 2011; Shalit-Kaneh et al., 2018). In addition to transcriptional regulation, coordinated protein–protein interactions among circadian clock components add complexity that precisely adjusts circadian rhythms and output pathways (Para et al., 2007; Helfer et al., 2011; Lau et al., 2011). Here, we showed that the core Arabidopsis clock proteins PRR5, TOC1, and ELF3 interact with each other and cooperatively regulate PIF4 transcription to repress hypocotyl elongation during the night. The circadian-clock-mediated suppression of PIF4 expression may allow plants to adjust their growth according to photoperiod. Under long-day or ND conditions, PIF4 protein stability and activity are suppressed by light-activated phyB during the day (Huq and Quail, 2002), whereas PIF4 expression is repressed by the PRR–EC complex throughout the night, ensuring that there is little hypocotyl elongation under these conditions. Under short-day or extended dark conditions, however, the suppression of PIF4 expression mediated by PRR–EC is alleviated late in the night, resulting in greater hypocotyl elongation around the dark-to-light transition (Zhu et al., 2016; Li et al., 2020; Hwang et al., 2021). Measuring the length of the night during one diurnal cycle is important for proper hypocotyl elongation, with the repressive transcriptional activity of the PRR–EC complex likely relevant for a certain time period after dusk. The nighttime inhibition of elongation is important for preventing excessive growth and maintaining energy conservation in the dark.

PRR5, TOC1, and ELF3 are largely interdependent in the control of hypocotyl elongation, although they may also have independent functions to some extent. Overexpression of PRR5 or TOC1 resulted in shorter hypocotyls with lower PIF4 levels, but this effect was significantly impaired in the elf3-1 mutant background. Similarly, the effect of ELF3 overexpression on the repression of hypocotyl elongation was diminished in the prr-q mutant background. Co-expression of PRRs and ELF3 enabled the synergistic repression of PIF4 expression compared with the expression of PRRs or ELF3 alone. We demonstrated here that the interdependence among PRR5, TOC1, and EC results from recruitment of the SWR1 complex, which is responsible for H2A.Z exchange at PIF4 chromatin. Recruitment of the SWR1 complex is further enhanced when the EC and PRRs co-exist; consistent with this notion, genes targeted by both the EC and PRRs have the highest H2A.Z enrichment across their loci. Indeed, the function of the PRR–EC complex in hypocotyl elongation depends on the SWR1 complex, although we cannot rule out the possibility that it may also involve additional regulatory mechanisms such as chromatin modifications and RNA-dependent gene control.

Notably, H2A.Z deposition is reversibly regulated in response to environmental fluctuations. For example, the SWR1 complex modulates global H2A.Z deposition, including at the key signaling hub PIF4, in response to low ambient temperatures (Kumar and Wigge 2010). By contrast, the INOSITOL REQUIRING 80 (INO80) chromatin remodeling complex mediates H2A.Z eviction at the PIF4 locus at high temperatures (Xue et al., 2021), ensuring balanced regulation by H2A.Z-containing nucleosomes as a function of ambient temperatures. Similarly, given that H2A.Z deposition is diurnally controlled, specifically at EC-target genes, the H2A.Z eviction mechanism might also be diurnally activated by other clock components. In addition, because H2A.Z dynamics play an important role in environmental acclimation in plants, the circadian clock may participate in diverse aspects of plant–environment interactions in an H2A.Z-dependent manner. Indeed, the EC and PRRs are involved in thermomorphogenesis (Thines and Harmon, 2010; Nusinow et al., 2011; Ding et al., 2018; Zhang et al., 2021), shade avoidance (Zhang et al., 2020), and stress responses (Liu et al., 2013; Nakamichi et al., 2016), possibly through control of H2A.Z states.

In plants, the EC component ELF3 contains a prion-like domain (PrD) and serves as a thermosensor through temperature-dependent liquid–liquid phase separation (LLPS) (Jung et al., 2020). Given that several databases, including Prion-Like Amino Acid Composition (PLAAC; http://plaac.wi.mit.edu/), predict that PRR5, TOC1, and several SWR1 components likely have PrDs, the PRR–EC–SWR1 complex may undergo LLPS diurnally and establish a cellular reaction center for chromatin remodeling that optimizes plant growth and environmental acclimation. Future studies should explore the involvement of LLPS in integrating the circadian clock and chromatin modifications for the control of plant development.

Methods

Plant materials and growth conditions

The A. thaliana accession Columbia (Col-0) was used for all experiments. Plants were grown under ND conditions (12-h light/12-h dark) using cool-white fluorescent lamps (120 μmol photons m−2 s−1) at 22°C. The prr5-1, toc1-21, elf3-1, prr5-1 toc1-21, prr5-1 elf3-1, toc1-21 elf3-1, prr5-1 toc1-21 elf3-1, arp6-1, and sef-1 mutants were described previously (Deal et al., 2005; Ding et al., 2007; March-DíAz et al., 2007; Wang et al., 2010; Ezer et al., 2017; Li et al., 2020). The elf3-1 proELF3:ELF3-MYC, TMG-YFP (proTOC1:TOC1-YFP), PRR5-ox, TOC1-ox, and proPRR5:PRR5-eGFP transgenic lines were described previously (Más et al., 2003; Yamashino et al., 2003; Nakamichi et al., 2012; Ezer et al., 2017; Li et al., 2020).

Hypocotyl length measurement

Surface-sterilized seeds were sown on half-strength Murashige and Skoog (MS) medium supplemented with 1% (w/v) sucrose and solidified with 0.75% (w/v) agar. After stratification at 4°C for 3 days in the dark, seedlings were grown vertically at 22°C under ND conditions for 7 days or under continuous red light (5 μmol m−2 s−1) for 5 days. Seedlings were then photographed, and hypocotyl lengths were measured using ImageJ software (https://imagej.net/ij/).

For measurements of hypocotyl elongation rate, seedlings were germinated and grown at 22°C under ND conditions for 8 days and photographed every 3 h under near-infrared light (λ = 850 nm). Hypocotyl lengths were measured from the beginning of seed germination using ImageJ software, and elongation rates were calculated from length differences divided by the time interval (3 h) between consecutive images. For comparisons of daytime and nighttime elongation rates, the length differences between ZT0 and ZT12 (for daytime elongation) or between ZT12 and ZT24 (for nighttime elongation) were divided by the time interval (12 h).

Bioinformatics analysis

The genome browser at iRegNet (Shim et al., 2021) was used to analyze the binding of circadian components to the PIF4 locus (GEO: GSE36361, GSE35953, and GSE137264), as well as H2A.Z deposition in the elf3-1, arp6, and pie1 mutants (GEO: GSE109101, GSE108450, and GSE139459).

H2A.Z ChIP-seq data deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE109101 (Tong et al., 2020) were downloaded and used to examine the diurnal deposition of histone H2A.Z. ChIP-seq reads were trimmed using Trim Galore (v0.6.7) (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and mapped using Bowtie 2 (v2.4.5) (Langmead and Salzberg 2012) with default parameters. MACS2 (v2.2.7.1) (Zhang et al., 2008) was used to calculate the fold enrichment (immunoprecipitate/input) of mapped reads. Genes targeted by PRR5, TOC1, and/or ELF3 were identified from previous studies (Huang et al., 2012; Nakamichi et al., 2012; Tong et al., 2020). A set of random genes was selected from Araport11 annotated genes (www.araport.org). H2A.Z enrichment profiles of target genes were drawn using deepTools (Ramírez et al., 2016).

ChIP–qPCR analysis

ChIP assays were conducted using seedlings grown at 22°C under ND for 2 weeks. Whole seedlings were harvested at the indicated ZT time points and crosslinked in phosphate-buffered saline (pH 7.4) containing 1% (w/v) formaldehyde (BIOPURE, 4920F) for 15 min under vacuum. The seedlings were then frozen in liquid nitrogen and ground in nuclei lysis buffer (50 mM Tris–HCl [pH 8.0], 10 mM EDTA, 1% [w/v] SDS, 1 mM PMSF, and 1× protease inhibitor cocktail [Roche]), and solubilized chromatin was ultrasonicated at 4°C for 20 cycles (30 s on and 30 s off, full power) to approximately 500-bp fragments using a Bioruptor Pico (Diagenode). Fragmented chromatin was incubated overnight with anti-H2A.Z (Abcam, ab4174) or anti-MYC (Millipore, 05-724) antibodies at 4°C, followed by incubation with 25 μl of Protein A (Dynabeads, 10001D) or Protein G (Dynabeads, 10003D) magnetic beads. For immunoprecipitation, fragmented chromatin was incubated with 25 μl of anti-GFP magnetic agarose (ChromoTek, gtma-20) at 4°C overnight. Beads were washed with low-salt buffer (20 mM Tris–HCl [pH 8.0], 2 mM EDTA, 0.2% [w/v] SDS, 0.5% [v/v] Triton X-100, 150 mM NaCl, 1× protease inhibitor cocktail), high-salt buffer (20 mM Tris–HCl [pH 8.0], 2 mM EDTA, 0.2% [w/v] SDS, 0.5% [v/v] Triton X-100, 500 mM NaCl, 1× protease inhibitor cocktail), LiCl buffer (10 mM Tris–HCl [pH 8.0], 1 mM EDTA, 0.5% [v/v] Nonidet P-40, 0.5% [w/v] sodium deoxycholate, 250 mM LiCl, 1× protease inhibitor cocktail), and then twice with TE buffer (10 mM Tris–HCl [pH 8.0], 1 mM EDTA). Chromatin was eluted in 500 μl of elution buffer (1% [w/v] SDS, 0.1 M NaHCO3) and reverse crosslinked overnight at 65°C. After proteinase K treatment (Biopure, 7730P), DNA fragments were purified by phenol/chloroform extraction and quantified by qPCR.

RT–qPCR analysis

Seeds were sown on half-strength MS medium supplemented with 1% (w/v) sucrose and solidified with 0.75% (w/v) agar, then grown under ND conditions for 2 weeks. Whole seedlings were harvested at the indicated ZT time points. To extract total RNA, samples were ground in liquid nitrogen, and 1 ml of TransZol Up (TransGen Biotech, ET111-01) was mixed thoroughly with the ground tissue. After centrifugation at 12 000 g for 10 min at 4°C, the supernatant was transferred to a new Eppendorf tube, and 250 μl of chloroform was added. RNA in the aqueous phase was precipitated with isopropanol. The RNA pellet was washed twice with 70% (v/v) ethanol and dissolved in RNase-free deionized water. Two micrograms total RNA was treated with DNase I (New England Biolabs, M0303L), followed by reverse transcription using M-MLV reverse transcriptase (MG MED, MR01601). qPCR was performed in 96-well plates on a Step-One Plus Real-Time PCR System (Applied Biosystems) using gene-specific primers (Supplemental Table 2) and SYBR Master MIX (Enzynomics, RT501M). The expression of each gene was normalized to that of EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (eIF4a) (At3g13920). All RT–qPCR reactions were performed using cDNA synthesized from total RNA extracted from independent biological triplicates. Relative expression levels were calculated using the ΔΔCt method. The Ct value of each reaction was determined automatically by the system using default parameters. The specificity of RT–qPCR reactions was determined by melting curve analysis of the amplified products using the standard method installed in the system.

Transient expression assays using Arabidopsis protoplasts

For construction of effector plasmids, the full-length cDNAs of PRR5, TOC1, and ELF3 were cloned into pEarleygate201 and pEarleygate203 vectors via the Gibson assembly cloning method (Gibson et al., 2009). For construction of reporter plasmids, ∼2052 bp of promoter sequence from the PIF4 locus and the entire PIF4 genomic coding region were cloned into a modified pCAMBIA1305 vector containing a minimal cauliflower mosaic virus (CaMV) 35S promoter and the β-glucuronidase (GUS) gene. A plasmid with the firefly luciferase (LUC) gene driven by the 35S promoter was co-transfected as an internal control for quantification of transfection efficiency.

Protoplast isolation and DNA transfection were performed as described previously with minor modifications (Yoo et al., 2007). In brief, mesophyll protoplasts were isolated from 3-week-old Arabidopsis seedlings grown under ND conditions. Twenty micrograms of each effector, reporter, and internal control plasmid was co-transfected into 1.2 × 106 protoplasts using the polyethylene glycol–mediated transfection method. After 16 h of incubation in W5 medium in the dark at 22°C, GUS and LUC activities were measured at ZT16 using a Tristar2 LB942 Multimode Microplate Reader (Berthold Technologies).

Y2H assays

Y2H assays were performed using the BD Matchmaker system (Clontech). Full-length cDNAs or cDNA fragments of PRR5, TOC1, ELF3, ARP6, SEF, PIE1 (535–825 amino acids), and PIE1 (1670–1730 amino acids) were subcloned into the pGADT7 or pGBKT7 vector to generate GAL4 activation domain (AD) and GAL4 DNA-binding domain (BD) fusion constructs, respectively. Full-length GAL4 was expressed as a positive control. The GAL4 AD and BD constructs were co-transformed into the yeast (Saccharomyces cerevisiae) strain pJG69-4A harboring the lacZ and HIS3 reporter genes, and transformed cells were selected by growth on synthetic defined (SD) medium lacking leucine and tryptophan (SD/–Leu/–Trp). Protein–protein interaction was tested by growth on SD medium lacking Leu, Trp, histidine, and adenine (SD/–Leu/–Trp/–His/–Ade).

BiFC assays

For BiFC assays, the full-length coding sequences of PRR5 and TOC1 were cloned into the pSAT4-nEYFP-N1 vector in frame with the sequence encoding the N-terminal 173 amino acids of YFP (nYFP). The full-length ELF3 coding sequence was inserted into the pSAT4-cEYFP-C1-B (E3082) vector in frame with the sequence encoding the C-terminal 68 amino acids of YFP (cYFP). The resulting constructs were co-transfected with a nuclear marker (mCherry-NLS) into Arabidopsis protoplasts. After incubation for 16 h in the dark at 22°C, transfected protoplasts were observed using a CQ1 confocal quantitative image cytometer (Yokogawa).

CoIP assays using Arabidopsis protoplasts

For protein production in Arabidopsis protoplasts, the full-length coding sequences of PRR5, TOC1, ELF3, and PIE1 were cloned into pEarleygate201 (HA-tag), pEarleygate202 (FLAG-tag), or pEarleygate203 (MYC-tag) plasmids. Plasmids (60 μg) were individually transfected into 3.6 × 106 protoplasts via polyethylene glycol–mediated transfection. After incubation in the dark for 16–24 h, transfected protoplasts were harvested in cold IP lysis buffer (50 mM Tris–HCl [pH 7.5], 1 mM EDTA, 150 mM NaCl, 10% [v/v] glycerol, 0.5% [v/v] Triton X-100, 1× protease inhibitor cocktail) and incubated in ice for 15 min. Total lysates were briefly sonicated for nucleus disruption at 4°C for three cycles (30 s on and 30 s off). After centrifugation at 13 000 g for 20 min at 4°C, supernatants were incubated with either 25 μl of Pierce anti-hemagglutinin (HA) magnetic beads (Thermo, 88837) or anti-FLAG M2 magnetic beads (Sigma, M8823). After incubation overnight at 4°C, beads were washed three times with IP wash buffer (50 mM Tris–HCl [pH 7.5], 1 mM EDTA, 150 mM NaCl, 10% [v/v] glycerol, 0.1% [v/v] Triton X-100, 1× protease inhibitor cocktail). Immunoprecipitated fractions were eluted using acid elution buffer (100 mM glycine, pH 2.5) and subsequently treated with neutralization buffer (1.5 M Tris–HCl, pH 8.8). An adequate amount of 5× SDS loading buffer was added, and the mixture was boiled for 5 min at 100°C. The immunoprecipitates were analyzed by immunoblotting using an anti-HA antibody (Millipore, 05-904; 1:5000), anti-FLAG M2 antibody (Sigma, F1804; 1:2000), and anti-MYC antibody (Millipore, 05-724; 1:5000).

Split-luciferase assays

For split-luciferase assays, the full-length coding sequences of PRR5, TOC1, and ELF3 were cloned into the pGWB-nLUC (amino acids 1–416 of firefly luciferase) vector, and the full-length coding sequence of PIE1 was inserted into the pGWB-cLUC (amino acids 398–550 of firefly luciferase) vector. The resulting constructs were co-transfected into Arabidopsis protoplasts. A 35S:GUS plasmid was used as an internal control for measuring transfection efficiency. After incubation for 16–24 h in the dark at 22°C, protoplasts were resuspended in imaging solution (W5 medium containing 2 mM D-luciferin) for measurement of luciferase activity. Subsequently, protoplasts were lysed for GUS activity measurement.

Protein domain analysis

Protein sequences were downloaded from The Arabidopsis Information Resource (https://www.arabidopsis.org/). Protein sequences were submitted to the Batch CD-Search Tool on the NCBI portal for domain analysis. Proteins with putative prion-like domains were identified using PLAAC software (PLAAC; http://plaac.wi.mit.edu/).

Statistical analysis

Significant differences among multiple groups were analyzed using one-way ANOVA followed by Tukey’s test at p < 0.05. Significant differences between two groups were assessed by two-tailed Student’s t-test. Statistical tests were performed using the agricolae v.1.3-3 package in R. Statistical parameters, including the exact value of n, the definition of center, dispersion, and precision measures (mean ± SEM or SD), and statistical significance are reported in the figures and figure legends.

Funding

This work was supported by the Basic Science Research (NRF-2022R1A2B5B02001266 to P.J.S. and NRF-2023R1A2C3002386 to E.O.) and Basic Research Laboratory (NRF-2022R1A4A3024451 ) programs provided by the 10.13039/501100003725 National Research Foundation of Korea .

Author contributions

P.J.S. and E.O. conceived and designed the study. J.H.W., J.P., and H.G.L. performed genetic and molecular analyses. S.S. and H.L. performed bioinformatics analysis. P.J.S., E.O., and J.H.W. wrote the manuscript.

Supplemental information

Document S1. Supplemental Figures 1–13 and Supplemental Table 2

Supplemental Table 1. List of genes co-targeted by PRR5, TOC1, and EC

Document S2. Article plus supplemental information

Acknowledgments

We thank Dr. Lei Wang (Chinese Academy of Sciences, China) for providing toc1-21, prr5-1, toc1-21 prr5-1, toc1-21 elf3-1, prr5-1 elf3-1, toc1-21 prr5-1 elf3-1, and proPRR5:PRR5-GFP seeds. No conflict of interest is declared.

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information is available at Plant Communications Online.
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