PHYTOCHROME-INTERACTING FACTOR 7 and RELATIVE OF EARLY FLOWERING 6 act in shade avoidance memory in Arabidopsis

Cheng, Qican; Zeng, Yue; Huang, Sha; Yang, Chuanwei; Xie, Yu; Shen, Wen-Hui; Li, Lin

doi:10.1038/s41467-024-51834-4

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Published: 13 September 2024

PHYTOCHROME-INTERACTING FACTOR 7 and RELATIVE OF EARLY FLOWERING 6 act in shade avoidance memory in Arabidopsis

Qican Cheng¹,
Yue Zeng¹,
Sha Huang¹,
Chuanwei Yang²,
Yu Xie¹,
Wen-Hui Shen³ &
…
Lin Li ORCID: orcid.org/0000-0003-4840-5245¹

Nature Communications volume 15, Article number: 8032 (2024) Cite this article

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Abstract

Shade avoidance helps plants maximize their access to light for growth under crowding. It is unknown, however, whether a priming shade avoidance mechanism exists that allows plants to respond more effectively to successive shade conditions. Here, we show that the shade-intolerant plant Arabidopsis can remember a first experienced shade event and respond more efficiently to the next event on hypocotyl elongation. The transcriptional regulator PHYTOCHROME-INTERACTING FACTOR 7 (PIF7) and the histone H3K27-demethylase RELATIVE OF EARLY FLOWERING 6 (REF6) are identified as being required for this shade avoidance memory. RNA-sequencing analysis reveals that shade induction of shade-memory-related genes is impaired in the pif7 and ref6 mutants. Based on the analyses of enrichments of H3K27me3, REF6 and PIF7, we find that priming shade treatment induces PIF7 accumulation, which further recruits REF6 to demethylate H3K27me3 on the chromatin of certain shade-memory-related genes, leading to a state poised for their transcription. Upon a second shade treatment, enhanced shade-mediated inductions of these genes result in stronger hypocotyl growth responses. We conclude that the transcriptional memory mediated by epigenetic modification plays a key role in the ability of primed plants to remember previously experienced shade and acquire enhanced responses to recurring shade conditions.

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Introduction

Plant growth and crop production are strongly affected by environmental conditions. Plants can detect the proximity of other plants by perceiving a change in the ratio of red light to far-red (R/FR) light from neighbouring plants and trigger a series of changes in architecture and physiology; this phenomenon is called shade avoidance syndrome (SAS)¹. The phenotypes associated with SAS include increased elongation of internodes and/or petioles, reduced leaf growth, and increased apical dominance, which occur together as an attempt of plants to avoid being shaded². Prolonged exposure to shade evokes the acceleration of flowering³, seed set reduction, and susceptibility to herbivores^2,4.

In recent years, the molecular mechanisms underlying shade-avoidance responses have been elucidated, mostly through studies in Arabidopsis. A reduction in R/FR irradiance is perceived by phytochromes (e.g., phyB)⁵. Typically, a phytochrome molecule exists in one of the two conformations, Pr and Pfr, the latter being the active form. The absorption of R light converts Pr to Pfr, whereas FR absorption reverts Pfr to Pr. PHYTOCHROME-INTERACTION FACTORs (PIFs) are basic HLH transcription factors capable of promoting stem extension growth⁶. PIF7 is considered a master regulator of SAS, as evidenced by the strong shade-defective response of pif7 mutant plants^7,8. PIF7 is less vulnerable than PIF1/3/4/5 to the rapid turnover induced by the Pfr form of phyB. The activity of PIF7 is controlled by rapid dephosphorylation in response to shade^8,9. Due to the reversible interconversion between the two states of phytochromes according to the R/FR ratio, the effects of shade on downstream gene expression and the organ elongation growth rate are reversible^10,11. For example, the abundance of the PHYTOCHROME-INTERACTING FACTOR 3-LIKE 1 (PIL1) transcript increased 35-fold after 1 h of exposure to a low R/FR ratio and quickly decreased after a return to a high R/FR ratio¹². Accordingly, the growth rate of hypocotyls increases under low R/FR treatment and decreases after reversion to high R/FR¹⁰.

More recent studies have revealed that some chromatin modifiers are involved in SAS. It has been shown that PIF7 interacts with MRG1/MRG2, which recruits histone acetylases to perform histone acetylation and promote the expression of shade-responsive genes¹³. HDA9 cooperates with PIF4/7 to regulate shade/auxin-responsive genes in response to shade¹⁴. PIFs also act in modifying the epigenetic landscape of the histone variants H2A.Z and H3.3 via interactions with EIN6 ENHANCER (EEN) and ANTI-SILENCING FACTOR 1 (ASF1) in response to shade^9,15. Shade also induces the production of lncRNAs (long non-coding RNAs) involved in modulating SAS¹⁶.

While molecular memory mechanisms are involved in plant adaptation to diverse biotic and abiotic stresses¹⁷, thus far, it is unknown whether plants can remember the first experienced shade and change their growth strategy in response to the second shade exposure. Thus, our study aimed to explore the memory of the shade avoidance response of Arabidopsis seedlings. We found that Arabidopsis seedlings can remember 8 h of primed shade after 40 h recovery, triggering faster hypocotyl elongation when exposed to shade again. Based on the phenotypic responses of several examined Arabidopsis mutants, ref6 and pif7 were found to abolish shade memory. RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) is required for normal shade-induced hypocotyl elongation and is responsible for demethylating H3K27me3 on REF6-targeted shade-induced genes. During the priming shade, PIF7 physically interacts with REF6, which demethylates H3K27me3 on certain PIF7-targeted shade-induced genes. Although these genes return to lower levels of expression during the recovery stage, they are maintained in a poised state, making them ready for induction, which results in stronger shade induction when shade occurs again.

Results

Arabidopsis seedlings exhibit shade avoidance memory

Using the DynaPlant system¹⁸, we monitored the time-lapse growth rates of hypocotyl elongation of Arabidopsis seedlings during white light/shade transitions. We found that shade-induced high growth rates gradually decreased to low levels over approximately 8–9 h after the Col-0 seedlings being transferred to white light (Fig. 1a). To test whether plants can remember their first experienced shade (priming with 2/4/8/10 h of shade), we exposed them to a second shade (triggering) after 40 h of white light recovery (recovering), which allowed their growth rate to return completely to the baseline level before triggering. As shown in Fig. 1b, c, the growth rates in response to triggering shade were similar between naïve (N, not exposed to a priming shade) and 2 or 4 h shade-primed (P) seedlings. However, 8/10 h shade-primed seedlings grew significantly faster than naïve plants during triggering shade (Fig. 1d, e).

**Fig. 1: *Arabidopsis* seedlings exhibit a shade avoidance memory in indicated light condition.**

We also measured the final hypocotyl lengths of naïve and primed seedlings after 48 h of triggering shade. Consistent with the growth rates, 8/10 h but not 2/4 h shade-primed seedlings showed longer hypocotyls than naïve plants (Supplementary Fig. 1a). This difference in hypocotyl length could have been caused by the priming shade induced elongation and/or a faster growth during the triggering shade caused by the memory of the priming shade. We found that the hypocotyl grew approximately 300 μm after the 8 h of priming shade, which was much less than the difference between naïve and primed seedlings after triggering shade treatment, indicating that plants remember the priming shade to accelerate growth when exposed to shade again.

To test the effects of recovery duration on the memory of the 8 h of shade priming, we extended the recovery time from 40 h to 48 h or 72 h. As shown in Supplementary Fig. 1b, the difference between naïve and shade-primed seedlings decreased with increasing recovery time. The faster growth caused by the memory of 8 h priming shade was detectable after 24 h and 48 h triggering shade application (Supplementary Fig. 1c).

Taken together, the results showed that Arabidopsis seedlings could remember the experience of 8 h priming shade, leading to faster growth after 40 h of recover when the second triggering shade occurred. The treatment of seedlings shown in Supplementary Fig. 1d was applied to detected final hypocotyl length of shade avoidance memory (P growth condition).

To provide further evidence for bona fide shade avoidance memory, we investigated the shade memory phenotypes of pif7-1 and several histone modification mutants, including ref6-1 and ref6-3 (two allelic mutants of REF6/JMJ12, encoding H3K27me3 demethylase), elf6-3 (a mutant of ELF6/JMJ11, encoding H3K27me3 demethylase), jmj13g (a mutant of JMJ13, encoding H3K27me3 demethylase), ref6-1elf6-3, clf28 (a mutant of CLF, encoding H3K27me3 methyltransferase), atx1-2 (a mutant of ATX, encoding H3K4me3 methyltransferase), sdg25-1 (a mutant of SDG25, encoding H3K4me3 methyltransferase), sdg8 (a mutant of SDG8, encoding H3K36me3 methyltransferase), and atx1-2sdg25-1. The hypocotyl lengths of jmj13g, clf28, atx1-2, sdg25-1, sdg8, atx1-2sdg25-1 and proREF6::REF6-HA/ref6-1 were similar with the wild-type control Col-0 grown under P condition (Supplementary Fig. 1d–e). However, the hypocotyl lengths of shade avoidance memory were markedly decreased in pif7-1, ref6-1, ref6-3, elf6-3, and ref6-1elf6-3 (Supplementary Fig. 1e), indicating that shade avoidance memory is indeed gene function dependent.

PIF7 and REF6 are required for shade avoidance memory and the shade avoidance response

Among the mutants we screened for shade avoidance memory (Supplementary Fig. 1e), growth rate measurements also confirmed that the ref6-1, ref6-3, elf6-3, ref6-1elf6-3 and pif7-1 mutants but not the proREF6::REF6-HA/ref6-1, sdg25-1atx1-2, sdg25-1, atx1-2 and jmj13g mutants, displayed growth defects in phase II and phase IV under shade-primed (P) condition (Fig. 2a, Supplementary Fig. 2a). ELF6 and JMJ13 are close homologues of REF6. Mutant of ELF6 displayed a defective shade memory phenotype, while the mutant of JMJ13 did not. The growth defect in the shade memory phenotype was stronger in the double mutant ref6-1elf6-3 than that in any of the two single mutants, indicating the functional redundancy between REF6 and ELF6 in terms of shade memory. These results suggest that PIF7 and REF6/ELF6 are required for shade avoidance memory. We also monitored the growth rates of ref6-1, proREF6::REF6-HA/ref6-1, ref6-3, elf6-3, ref6-1elf6-3 and pif7-1 under naïve (N) condition. Unlike the growth rates under P condition, the shade growth defects of ref6-1, ref6-3, elf6-3, and ref6-1elf6-3 under N condition mainly occurred in phase IV but not phase II (Fig. 2b), indicating a possible difference in the mechanisms involved between the N and P conditions.

**Fig. 2: *PIF7* and *REF6* are required for shade avoidance memory and shade avoidance response.**

To check the growth rate in more detail, we measured the kinetics of growth decay after the priming shade treatment in Col-0, pif7-1, ref6-1 and ref6-3 mutants (Supplementary Fig. 2b, c). The mean time for extension growth to return to the level before shade treatment was approximately 8–9 h in Col-0, 4–5 h in ref6-3/ref6-1, and 3–4 h in pif7-1, suggesting that PIF7 and REF6 mediate the persistent effects of 8 h shade on hypocotyl elongation.

To further address the role of REF6/ELF6 in shade avoidance under N condition, 3-day-old seedlings grown under white light were transferred to shade for 4 d as normal conditions for the detection of shade-induced hypocotyl elongation. We found that ref6-1, ref6-3, elf6-3, and ref6-1elf6-3 exhibited markedly attenuated responses to shade compared with Col-0 (Supplementary Fig. 2d). The attenuated hypocotyl length of the ref6-1 mutant could be rescued by proREF6::REF6-HA complementation (Supplementary Fig. 2d). In contrast, the seedlings of sdg25-1atx1-2, sdg25-1, atx1-2 and jmj13g displayed the similar shade-induced hypocotyl elongation with Col-0, indicating that these genes may play no role on shade response and shade memory (Supplementary Fig. 2d).

To investigate the genetic relationships between REF6/ELF6 and PIF7/phyB, we generated ref6-1pif7-1, ref6-3pif7-1, elf6-3pif7-1, ref6-1phyB-9, and elf6-3phyB-9. Under N conditions, the hypocotyl lengths of ref6-1pif7-1, ref6-3pif7-1 and elf6-3pif7-1 were closer to that of pif7-1 (Supplementary Fig. 2d). Under white light conditions, loss of function of REF6/ELF6 could partially repress the long-hypocotyl phenotype of phyB-9 (Supplementary Fig. 2e), indicating that REF6 functions downstream of phyB. These results suggest that REF6 and ELF6 are involved in phyB-PIF7-modulated shade-induced hypocotyl elongation.

REF6 demethylates H3K27me3 on late shade-induced genes

Given the finding that the growth defects of ref6-1 and ref6-3 occur relatively late (phase IV) under N conditions, we wondered whether REF6 differentially regulates early and late shade-responsive genes. Exploiting a previously published RNA-sequencing dataset (GSE59722) that includes data profiles of both 1 h and 7 h shade treatments, we first reanalysed the differentially expressed genes (DEGs) with cut-off FC > 2 and FDR < 0.05, and we identified 1 h shade-induced genes (162) and 7 h shade-induced genes (739) (Fig. 3a, Supplementary Data 1). We then searched for their overlaps with genes bound by REF6 (2836 genes, GSE65329¹⁹). A higher number of REF6-bound genes were found overlapped with 7 h than 1 h shade-induced genes (Fig. 3a, Supplementary Data 2). These genes were enriched in growth related Gene Ontology (GO) categories, including hormone-mediated signalling pathway, developmental growth and cell wall biogenesis or organization (Fig. 3b, Supplementary Data 3).

**Fig. 3: REF6 maintains low H3K27me3 levels on late shade-induced genes.**

Moreover, in Col-0 grown under white light, the 7 h shade-induced genes displayed a drastically lower basal-level of H3K27me3 than the control reference genes (Fig. 3c). The basal-level of H3K27me3 in the 7 h shade-induced genes was higher in ref6-1 than in Col-0 based on GSE65329¹⁹ (Fig. 3d). This elevated H3K27me3 in ref6-1 was much more pronounced on the REF6-bound 7 h shade-induced genes (Fig. 3e). Moreover, we obtained similar results using other published RNA-sequence datasets with 1 h (GSE210807¹⁶) and 6 h (GSE226205²⁰) shade treatments (Supplementary Fig. 3).

To verify and extend the above observation, we performed quantitative PCR analyses on some selected genes. Based on the overlap between REF6-bound genes and 7 h shade-induced genes, we chose to analyse LNG2, EXPA11, XTH22 and XTH23 in Col-0 and ref6-1 at 1 h and 8 h of shade treatment. These genes have been reported to be involved in hypocotyl elongation and/or shade avoidance^{21,22,23,24,25}. Our RT‒qPCR data revealed that in Col-0, the transcription levels of these genes were induced significantly at 8 h but not at 1 h of shade treatment (Fig. 3f). In ref6-1, the expression levels of these genes were all reduced (Fig. 3f).

To investigate whether an increased H3K27me3 level could be associated with reduced expression levels of shade-induced genes in ref6-1, we carried out chromatin immunoprecipitation coupled with quantitative PCR analyses (ChIP-qPCR) with anti-H3K27me3 antibody. Our results showed that the H3K27me3 levels at LNG2, EXPA11, XTH22 and XTH23 did not change significantly upon shade treatment but were clearly increased in ref6-1 under all the studied conditions (Fig. 3g). These results indicated that, although shade barely influences the level of H3K27me3, basal levels of H3K7me3 are indeed determinant for transcription levels of shade-induced genes.

The expressions of these late shade-induced genes corelated with growth defect of ref6 in phase IV under N condition. However, this category of gene cannot explain the growth defect in phase II under P condition, indicating there may be an alternative regulatory mechanism.

Shade barely affects global genome-wide binding of REF6

The REF6 gene itself was not responsive to shade, as evidenced by its relatively constant transcript and protein levels in our analyses (Supplementary Fig. 4a, b).

To test whether shade affects genome-wide binding of REF6, we performed ChIP-seq analysis using the proREF6::REF6-HA/ref6-1 seedlings grown under N_WL and N_SH 8 h with an anti-HA antibody. A total of 7726 and 7205 peaks covering 7026 and 6702 genes (overlapping between two replicates, FC > 2 and q < 0.05) were bound by REF6-HA under N_WL and N_SH 8 h, respectively (Supplementary Data 4). These identified REF6-HA-bound genes were largely overlapped with the previously published REF6-HA-bound genes¹⁹ (Supplementary Fig. 4c). And the top enriched-motif bound by REF6-HA was CTCTGTTT, similar to that previously reported²⁶ (Supplementary Fig. 4d). Moreover, the results shown in Fig. 3a and Supplementary Fig. 3a were further supported by our data obtained under N_WL and N_SH 8 h, showing that there were more REF6-HA-bound genes associated with late then early shade-induced genes (Supplementary Fig. 4e, f). Remarkably, we found that the enrichment patterns of REF6-HA between N_WL and N_SH 8 h were highly correlated and overlapped with each other (Fig. 4a–c). We further examined the enrichment of REF6-HA at LNG2, EXPA11, XTH22 and XTH23. Consistent with H3K27me3 profiles (Fig. 3g), the enrichments of REF6-HA were barely affected by shade treatment at these examined genes (Fig. 4d, e). Taken together, these results indicate that shade does not affect obvious global genome-wide binding of REF6-HA.

**Fig. 4: Shade barely affects global genome-wide binding of REF6.**

REF6 and PIF7 regulate the expression of shade-memory-related genes

If REF6 participates in SAS only for maintaining the induction potential of shade-induced genes, the REF6 loss-of-function mutant should display the same shade avoidance memory phenotype as Col-0 under P condition. However, both ref6-1 and ref6-3 showed defective memory phenotypes, and the growth rate patterns of ref6-1 and ref6-3 were different between the N and P conditions. This suggests that REF6 has additional roles in shade avoidance memory.

According to the shade memory phenotype, plants acclimatized to shade respond to the triggering shade more strongly than non-acclimatized plants. Therefore, we conducted RNA sequencing to identify shade-memory-related genes whose inductions were enhanced in primed plants compared to naïve plants in response to triggering shade (Supplementary Fig. 5a). GO analysis of shade-induced genes, identified from both naïve and primed plants with the cut-offs FC > 1.5 and P < 0.05, indicated that “shade avoidance”, “response to auxin”, and “response to red or far-red light” are highly enriched terms (Supplementary Fig. 5a–c, Supplementary Data 5), which is consistent with previously published datasets^15,27. The inductions of shade-induced genes identified in naïve plants were found reduced in both pif7-1 and ref6-1 under N condition, and the reductions were less severe under P condition (Supplementary Fig. 5d). Vice versa, the inductions of shade-induced genes identified in primed plants were reduced in both pif7-1 and ref6-1 more severely under P condition than N condition (Supplementary Fig. 5e). These results indicate that shade-induced genes were different between naïve and primed plants, which contributed the to the shade deficits under N condition and shade memory deficits under P condition, respectively.

Then, we chose genes with increased shade-induction (1478, FC > 1.2) under P compared to N conditions and referred them as shade-memory-related genes (Fig. 5a, Supplementary Data 6). The enriched growth-related GO terms (auxin-activated signalling pathway, growth, cell wall organization) in these shade-memory-related genes (Fig. 5b, Supplementary Data 7) supported the increased growth rates under P conditions. These results suggest that transcriptional memory²⁸ participates in shade avoidance memory.

**Fig. 5: Transcriptome profiling reveals shade-memory-related genes.**

Moreover, the shade induction of these shade-memory-related genes was impaired in primed ref6-1 and more severely in primed pif7-1 (Fig. 5a, c), which is consistent with the shade memory defective phenotypes of these mutants. To further explore whether the effect of REF6 on shade avoidance memory is associated with PIF7, we used mfuzz clustering of differentially expressed shade-memory-related genes in Col-0, pif7-1 and ref6-1. An optimal number of clusters (four) were identified by using Pearson correlation based on levels of gene expression. We found two clusters (C1:401, C3:443 genes) (Supplementary Data 8) comprising 844 downregulated genes in both pif7-1 and ref6-1 (Fig. 5c), in which growth terms were enriched, such as cell wall organization, response to hormone, and growth. The previous examined genes LNG2, EXPA11, XTH22 and XTH23 were absent from this list of 844 genes. Interestingly, five other genes (IAA19. BGL1. GH3.6. PRE1 and YUCCA2) previously reported as playing a role on hypocotyl elongation and/or shade avoidance^{7,29,30,31,32,33,34} were among the identified genes. The triggering-shade induction of IAA19, BGL1, GH3.6, PRE1 and YUCCA2 in plants grown under N and P conditions were further confirmed by RT‒qPCR analysis (Fig. 5d and Supplementary Fig. 5f). The decreased triggering-shade induction of these genes in primed pif7-1 and ref6-1 as well as in primed elf6-3 was also confirmed (Fig. 5d and Supplementary Fig. 5f–g). Together, these results establish that PIF7 and REF6/ELF6 participate in regulating the expression of shade-memory-related genes.

Demethylation of H3K27me3 at certain shade-memory-related genes is mediated by PIF7 and REF6

Next, we re-analysed our REF6-HA ChIP-seq data by specifically focusing on the 844 shade-memory-related genes coregulated by PIF7 and REF6. Interestingly, these genes showed a moderate but statistically significant higher enrichment of REF6-HA under N_SH 8 h than under N_WL conditions (Fig. 6a). To further explore REF6-HA binding at shade-memory-related genes, we identified shade-increased REF6-HA-binding genes by using lower FC (N_SH 8 h /N_WL > 1.2. Supplementary Data 9) and compared them with the 844 shade-memory-related genes and PIF7-bound genes (GSE156584³⁵). This led to the identification of 17 common genes and 17 shade-memory-related genes showing shade-increased enrichment of REF6-HA but not PIF7-bound (Fig. 6b). Growth-related GO terms were enriched mainly in the REF6 and PIF7 co-targeted genes (Fig. 6c). The shade-increased enrichment of REF6-HA was found greater for these REF6 and PIF7 co-targeted genes (Fig. 6d).

We were curious to know why the enrichment of REF6-HA increased on a number of PIF7-targeted genes under shade. First, we confirmed the binding of PIF7 at IAA19, BGL1, GH3.6, PRE1 and YUCCA2 by ChIP‒qPCR analysis (Fig. 6e and Supplementary Fig. 6b–c). Then, we checked the H3K27me3 levels on PIF7-targeted genes in white light-grown naïve plants (N_WL), 1 h shade-treated naïve plants (N_SH 1 h), 8 h shade-treated naïve plants (N_SH 8 h), recovering primed plants (P_WL), and 1 h shade-treated primed plants (P_SH 1 h) (the treatment of samples was shown in Supplementary Fig. 6a). The transcriptional level of PP2AA3 is not regulated by shade and is unchanged in ref6-1 and pif7-1 according to our qRT-PCR results (Supplementary Fig. 6d), and there is H3K27me3 mark at the PP2AA3 locus based on the previously published H3K27me3 ChIP-seq data (GSE18129³⁶). Therefore, we chose PP2AA3 as a negative control for H3K27me3 in our ChIP-qPCR analysis. Remarkably, 1 h of shade did not change the level of H3K27me3 on our examined genes in either naïve or primed plants (N_WL vs. N_SH 1 h, P_WL vs. P_SH 1 h), but 8 h of shade decreased H3K27me3 levels (N_WL vs. N_SH 8 h). Low levels of H3K27me3 were maintained during the recovery time (P_WL) and 1 h of triggering shade time (P_SH 1 h) (Fig. 6f and Supplementary Fig. 6e). The level of H3K27me3 on PP2AA3 was stable under all our tested conditions and in all our studied genotypes (Supplementary Fig. 6e). As expected, the reductions in H3K27me3 caused by 8 h of shade were absent in pif7-1 and ref6-1 (Fig. 6f), suggesting that both PIF7 and REF6 are required for the observed changes of H3K27me3 at these examined shade-memory-related genes.

Enrichment of REF6 at certain shade-memory-related genes is dependent on PIF7

Next, we asked the question whether REF6 enrichment at shade-memory-related genes dependent on PIF7. For this, we investigated the enrichment of REF6-HA in the pif7-1 background under N_SH 8 h by ChIP-seq analysis. A total of 5956 peaks covering 5687 genes were identified to be associated with REF6-HA binding in pif7-1 (overlapping between two replicates, FC > 2 and q < 0.05, Supplementary Data 10). There were 4760 overlapped REF6-HA binding genes between ref6-1 and pif7-1 background (Fig. 7a). The enrichment patterns of REF6-HA on these genes were highly correlated in pif7-1 as compared to ref6-1 (Fig. 7b–c). To further explore the effect of PIF7 on REF6-HA binding, we identified increased and decreased REF6-HA-binding genes in pif7-1 by using FC > 1.2 (Supplementary Data 11). Growth related GO terms, such as response to light stimuli, response to hormone, were enriched in decreased REF6-HA-binding genes in pif7-1 (Fig. 7d), in which there were 312 PIF7 bound genes (Fig. 7e). A reduction of REF6-HA enrichment in pif7-1 as compared to ref6-1 was observed on the REF6 and PIF7 co-targeted genes identified in Fig. 6b (Fig. 7f), suggesting a pivotal role of PIF7 in REF6 enrichment on certain shade-memory-related genes.

We further performed ChIP‒qPCR analysis to verify the binding of REF6 at IAA19, BGL1, GH3.6, PRE1 and YUCCA2 in white light-grown naïve plants (N_WL), 1 h shade-treated naïve plants (N_SH 1 h), 8 h shade-treated naïve plants (N_SH 8 h), recovering primed plants (P_WL), and 1 h shade-treated primed plants (P_SH 1 h). We found that the enrichment of REF6-HA increased after 8 h of shade treatment (N_WL vs. N_SH 8 h) and this increase was maintained after 40 h recovering and 1 h of triggering shade (N_SH 8 h vs. P_WL and P_WL vs. P_SH 1 h) (Fig. 7g and Supplementary Fig. 7a). Interestingly, in pif7-1, the enrichment of REF6-HA remained low at all our studied treatment conditions (Fig. 7g and Supplementary Fig. 7a). Together with the ChIP-seq data, these ChIP-qPCR results support that the shade-induced enrichment of REF6 on certain shade-memory-related genes was dependent on PIF7.

Then we questioned whether the expression of PIF7 and REF6 disturbed each other. We analysed their transcript and/or protein level changes in pif7-1 and ref6-1 during the first and second light transitions. REF6 levels were quite stable and barely affected in pif7-1 (Supplementary Fig. 7b–d). The transcript level of PIF7 was repressed by shade in Col-0 as well as in ref6-1 (Supplementary Fig. 7e). While dephosphorylated PIF7 accumulated with shade treatment (Supplementary Fig. 7f), which is consistent with previous reports^8,37. These results indicate that the expression of PIF7 does not depend on the function of REF6 and vice versa.

PIF7 binds REF6

Next, we tested the protein‒protein interaction between PIF7 and REF6. We first used a luciferase complementation imaging (LCI) assay. As shown in Fig. 8a, nLUC-tagged PIF7 could interact with the cLUC-tagged C-terminus of REF6 when it was transiently expressed in Nicotiana benthamiana leaf cells. We successfully expressed SUMO-His-PIF7 and GST-REF6C, but failed to express GST-REF6 in E. coli. In an in vitro pull-down assay, SUMO-His-PIF7 proteins purified from E. coli were bound by GST-REF6C proteins purified from E. coli but not GST alone (Fig. 8b). To further examine the interaction between REF6 and PIF7 in vivo, we crossed the proREF6::REF6-HA/ref6-1 line with the 35S::PIF7-Flash (9 × Myc-6 × His-3 × Flag) line and performed co-immunoprecipitation (Co-IP) assays. As shown in Fig. 8c, the dephosphorylated, but not the phosphorylated, PIF7-Flash extracted from proREF6::REF6-HA/ref6-1*35S::PIF7-Flash could be precipitated by REF6-HA using an HA antibody. Since the dephosphorylated PIF7 protein accumulated with shade treatment, we further explored the interaction strength of REF6 and PIF7 over the time course of shade treatment. As expected, we found that more REF6-HA formed protein complexes with cumulative dephosphorylated PIF7–Flash (Fig. 8d). This finding suggests that PIF7 recruited more REF6 to participate in the removal of H3K27me3 on shade-memory-related genes during 8 h of priming shade treatment.

**Fig. 8: PIF7 physically interacts with REF6 to contribute to shade avoidance memory.**

Because the C-terminus of REF6 is highly homologous with that of ELF6 (Supplementary Fig. 8a), we also verified and confirmed the interaction between PIF7 and the C-terminus of ELF6 in LCI and in vitro GST pull-down assays (Supplementary Fig. 8b, c).

We further analysed the genetic relationship between PIF7 and REF6/ELF6 in shade memory determinacy. The primed shade-induced hypocotyl elongation disappeared in the double mutants pif7-1ref6-1, pif7-1ref6-3 and pif7-1elf6-3, which behaved more like pif7-1 (Fig. 8e, and Supplementary Fig. 8d). Growth rate measurements confirmed that the pif7-1ref6-1, pif7-1ref6-3 and pif7-1elf6-3 mutants displayed shade avoidance memory defects resembling those of pif7-1 (Fig. 2a, Fig. 8f, Supplementary Fig. 8e). These results suggest that PIF7 and REF6/ELF6 are required for shade avoidance memory.

Taken together, our results suggest that under prolonged shade, accumulated PIF7 can recruit REF6/ELF6, which reduces the level of H3K27me3 in shade-memory-related genes (Fig. 8g). These genes become more inclined to be induced in primed plants than in naïve plants, contributing to faster hypocotyl elongation under recurring shade (Fig. 8g).

Discussion

In nature, environmental changes, particularly stressful abiotic conditions, are often chronic or recurring. The initial response may be different from the long-term response and may prime a plant to respond differently to recurrence of the same condition. Although shade is not an extreme or harmful stress, plants have evolved sophisticated ways to acclimate or adjust their growth patterns according to the environmental conditions¹. Shade-intolerant plants, such as Arabidopsis, are able to quickly change gene expression programmes within minutes after the onset of shade conditions¹². With time, the transcriptional response diverges²⁷. Most gene expression changes can be reversed after the seedlings are returned to white light. The distinct expression patterns were observed between naïve and primed plants under the same triggering condition. These altered gene expression responses that occur upon repeated exposure to a condition have been termed transcriptional memory^38,39,40,41.

In the current study, we identified shade induced transcriptional memory. The H3K27me3 mark negatively regulates gene expression⁴² and a low level of H3K27me3 is a prerequisite for shade induction by LNG2, EXPA11, XTH22 and XTH23 (Fig. 3). Our work indicates that REF6 is responsible for maintaining the potential of certain REF6-targeted genes to be induced by shade. A similar mechanism was also reported under warm temperatures³⁶. Prolonged shade-induced genes include more REF6-targeted genes, which explains why the effect of REF6 on hypocotyl elongation mainly occurred at the late stage (Phase IV) under N condition (Fig. 2b, Supplementary Fig. 4g).

Meanwhile, shade led to a significant decrease in H3K27me3 levels in PRE1, YUCCA2, IAA19, BGL1 and GH3.6, accompanied by increased enrichment of REF6 after 8 h of priming shade treatment (Fig. 6). In addition to self-targeting, REF6 can be recruited to PIF7’s targets (Figs. 7, 8). These genes maintain lower levels of H3K27me3 during recovery compared to those in a plant in the naïve state. Although these changes are PIF7 dependent, PIF7 dissociates from these loci during recovery. A low level of H3K27me3 is retained to mediate transcriptional memory in the absence of ongoing transcription. When primed plants face shade again, the induction of these genes becomes stronger and occurs earlier (Fig. 8g).

Together, the results of our study reveal the molecular mechanism by which REF6 participates in the transcriptional response to prolonged shade and recurring shade and highlight the importance of the cooperation of epigenetic factors and transcription factors in regulating gene expression and environmental responses.

Within the same generation, the effects of priming can be maintained /remembered for a few days or weeks, suggesting that information can be stored during priming. This somatic transcriptional memory has been reported for drought response, salt response, development and differentiation, and so on. For example, the Δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) gene shows transcriptional memory (enhanced reinduction) in response to salt stress. Several epigenetic modifications have been implicated in stress memory after priming, such as histone H3K4 hypermethylation, histone H3K27 methylation, and nucleosome occupancy^39,43. The maintenance of a high level of H3K4me3 and stalled Ser5P RNA Polymerase II (Ser5p Po lI) at stress memory genes helps plants remember experienced dehydration stress⁴⁴. Heat acclimation induces sustained H3K27me3 demethylation at HEAT SHOCK PROTEIN22 (HSP22) and HSP17.6 C loci, poising the HSP genes for subsequent activation⁴⁵. In mouse TT2 embryonic stem cells (ESCs), CpG island (CGI)–binding protein BEND3 is required for association with Polycomb repressive complex 2 (PRC2) at bivalent genes, playing a reining function in maintaining high levels of H3K27me3 at those bivalent genes in ESCs to prevent their premature activation in the forthcoming developmental stage⁴⁶. In fact, 36 years ago, persistent effects of longer periods of shade or FR treatment on elongation growth were first reported after the plants were returned to white light⁴⁷. Later, we have learned more about the link between photoreceptor excitation and changes in plant architecture, especially in hypocotyl elongation, in Arabidopsis. A longer duration of shade triggers more downstream events. In the present study, we identified H3K27me3 modifications and found that PIF7 and REF6 are involved in shade-induced transcriptional memory. Epigenetic changes occur during prolonged shade treatment and trigger extension growth when the seedlings are facing shade again. Whether and how other epigenetic mechanisms are linked to shade-induced transcriptional memory will be an interesting topic for further study.

The removal of H3K27me3 is catalysed by JmjC ___domain-containing histone demethylases, including in REF6, ELF6 and JUMONJI 13 (JMJ13)^48,49,50. A previous report suggested that REF6 and ELF6 are necessary for heat acclimation⁴⁵. Both the ref6 and elf6 single mutants displayed similar shade memory defects in terms of growth rate and hypocotyl length (Figs. 1g, 2a), while JMJ13-defective mutant displayed no such defects (Supplementary Fig. 2d). The ref6-1elf6-3 showed slightly enhanced shade memory defects in hypocotyl length, suggesting that REF6 and ELF6 are partially redundant in shade memory. Due to their conserved C-terminus, both REF6 and ELF6 can be recruited by PIF7 (Fig. 8a–d and Supplementary Fig. 8) to certain shade- memory-related genes and regulate their expression in response to recurring shade (Supplementary Fig. 5g). This finding is comparable to a previous report showing that BES1 recruits REF6 and ELF6 to regulate target gene expression and coordinate the BR response⁵¹. It has also been reported that REF6 and ELF6 regulate the removal of H3K27me3 at different genomic loci due to their preference for binding motifs, which is supported by the distinct roles of REF6 and ELF6 in the maintenance of plant genome integrity⁵². However, due to the conserved motif, PIF7 can recruit both REF6 and ELF6, which have partially redundant functions in the response to recurring shade. The detailed features of this redundancy and differences among these three enzymes warrant more research.

Plants can remember past environmental conditions to better prepare for recurring stress⁵³. The duration of the memory varies depending on the type of environmental exposure. Here, we found that shade-induced memories are short-term memories. The effect of priming shade decreased with the extension of recovery time (Supplementary Fig. 1b). The length of the priming shade required for memory is related to the speed of H3K27me3 removal by demethylases. The recovery time may be dependent on the speed of writing of H3K27me3 and other epigenetic modifications. Heat induces transmitted phenotypes via a coordinated epigenetic network involving histone demethylases, transcription factors, and tasiRNAs, ensuring reproductive success and transgenerational stress adaptation⁵⁴. How the memory of shade is established and transmitted to progeny and whether phenotypes are affected remain largely unknown. These studies will further elucidate the mechanisms of plant adaptation and memory.

Methods

Plant materials and growth conditions

All Arabidopsis (Arabidopsis thaliana) plants used were in the Col-0 background. The information of mutants used in this study have been previously described: pif7-1⁸, ref6-1⁵⁵, ref6-3⁵⁵, elf6-3⁵⁵, ref6-1elf6-3⁵⁵, proREF6::REF6-HA/ref6-1⁵⁵, clf28, sdg25-1⁵⁶, atx1-2⁵⁶, atx1-2sdg25-1⁵⁶, sdg8, jmj13g³⁶, phyB-9 and 35S::PIF7-Flash⁸. The double mutants pif7-1ref6-1, pif7-1ref6-3, pif7-1elf6-3, phyB-9ref6-1, phyB-9elf6-3 proREF6::REF6-HA/pif7-1 and proREF6::REF6-HA/ref6-1*35S::PIF7-Flash were generated by genetic crossing and verified using phenotypic inspection, PCR genotyping, and/or sequencing.

For phenotypic analysis, seeds were germinated on plates containing 1/2 MS medium with 1% agar (Sangon, Shanghai, China) and without sucrose. After 5 days stratification, the plates were incubated in growth chambers under continuous white light (R, ~30 μmol m-2 s-1; B, ~20 μmol m-2 s-1; FR, ~8 μmol m-2 s-1) and then transferred to shade (R, ~30 μmol m-2 s-1; B, ~20 μmol m-2 s-1; FR, ~65 μmol m-2 s-1) at 22 °C. Nicotiana benthamiana plants were grown at 26 °C under long-day conditions with 16 h of light.

Hypocotyl measurement

The kinetics of hypocotyl growth were measured by a commercial high-throughput imaging platform, DynaPlant® (Microlens Technology, Beijing, http://www.dynaplant.cn/en). Seedlings for kinetic measurements were sown on 1/2 MS medium containing 2% phytagel (Solarbio, P8170) and grown under N (4-day-old seedlings + 8 h white light + 40 h recovering white light + 8 h triggering shade) and P (4-day-old seedlings + 8 h priming shade + 40 h recovering white light + 8 h triggering shade) conditions. The images of hypocotyl growth were captured by the DynaPlant® platform once every 15 min for each seedling with a physical resolution of 1.2 μm per pixel. The lengths of new hypocotyl growth in the time-series images were quantified by DynaPlant Analysis software, which was provided by the instrument manufacturer. The values shown indicate the means with SEM.

Quantitative measurements of hypocotyl lengths were performed on scanned images of seedlings using ImageJ software. At least 16 seedlings were used per treatment or genotype.

RNA-seq analysis

For Fig. 3a, differential expression analysis was performed using DESeq2 with |log₂foldchange | > log₂(2) and FDR < 0.05 using a web-based tool (http://bioinformatics.sdstate.edu/idep/). The raw RNA-seq data were obtained from GSE59722. For Fig. 5, seedlings were grown under N (4-day-old seedlings + 8 h white light + 40 h recovering white light + 1 h triggering shade) and P (4-day-old seedlings + 8 h priming shade + 40 h recovering white light + 1 h triggering shade) conditions. Two biological replicates were prepared for each genotype of the plants grown under light and shade conditions. Total RNA was extracted from snap-frozen tissues using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). RNA libraries were constructed and sequenced using Majorbio (http://www.majorbio.com/). Differential expression analysis was performed using DESeq2 with |log₂foldchange | > log₂(1.5) and P < 0.05. The heatmap of shade memory-related genes was generated by gene expression pattern clustering analysis using Majorbio (http://www.majorbio.com/). We chose genes with increased shade-induction (FC > 1.2) under P compared to N conditions in 1876 P_shade-induced genes and referred them as shade-memory-related genes. GO term enrichment analysis was conducted by the web-based tool DAVID.

Quantitative RT‒PCR analysis

Approximately 100 mg of seedlings grown on 1/2 MS medium supplemented with 1% agar under different light conditions was collected in tubes, frozen in liquid nitrogen, and ground to a fine powder. Three biological replicates were prepared for each genotype of the plants grown under white light and shade conditions (N, 4-day-old seedlings + 8 h white light + 40 h recovering white light + 1 h triggering shade; P, 4-day-old seedlings + 8 h priming shade + 40 h recovering white light + 1 h triggering shade). Total RNA was extracted using a TRIzol kit (Promega, USA). Two micrograms of total RNA were reverse transcribed using a First Strand cDNA Synthesis Kit (TIANGEN, China) according to the manufacturer’s instructions. The cDNAs were then subjected to real-time qPCR using a CFX Connect Real-Time System (Bio-Rad, USA) and SYBR Green qPCR Mix (Mei5 Biochem, China). Three biological replicates per sample were used for the qRT‒PCR analysis. The data are presented as the means with the SD of three biological replicates normalized to the expression of the reference gene AT2G39960. The comparative ΔΔCt method was used to evaluate the relative quantities of each amplified product in the samples. The specificity of the qRT-PCRs was determined by melt curve analysis of the amplified products using the standard method. The primers used are listed in Supplementary Data 12.

ChIP‒qPCR assay

For H3K27me3 ChIP‒qPCR, Col-0, pif7-1, and ref6-1 seedlings were grown under white light and shade conditions (N, 4-day-old seedlings + 8 h white light + 40 h white light + 1 h and 8 h triggering shade; P, 4-day-old seedlings + 8 h priming shade + 40 h white light + 1 h and 8 h triggering shade). Three biological replicates were prepared for each genotype of plants grown under light and shade conditions. For HA ChIP‒qPCR, proREF6::REF6-HA/ref6-1 and proREF6::REF6-HA/pif7-1 seedlings were grown under white light and shade conditions (N, 4-day-old seedlings + 8 h white light + 40 h white light + 1 h and 8 h triggering shade; P, 4-day-old seedlings + 8 h priming shade + 40 h white light + 1 h triggering shade). Three biological replicates were prepared for each genotype of the plants grown under light and shade conditions. The seedlings were harvested and cross-linked for 15 min under vacuum in cross-linking buffer (extraction buffer 1 with 1% formaldehyde). Cross-linking was terminated with 125 mM glycine (pH 8.0) under vacuum for 5 min, and the seedlings were washed three times in double-distilled water and rapidly frozen. A bioruptor was used at high power with 15 30 s on /30 s off cycles until the average chromatin size was approximately 200-500 bp, followed by immunoprecipitation with an anti-HA (Lumiprobe sc-7392 X) or anti-H3K27me3 antibody (Millipore 07-449). RT‒qPCR was performed using a kit from Takara to determine the enrichment of immunoprecipitated DNA in the ChIP experiments. The TA3 retrotransposon (AT1G37110) and AT2G39960 were used as the negative control region for ChIP‒qPCR. The values in the graphs are the mean ± SD. At least three independent biological replicates were performed. Statistical significance was determined by one-way ANOVA with Tukey’s HSD test for multiple-pair comparisons. The primers used are listed in Supplementary Data 12.

ChIP-seq analysis

For REF6-HA binding ChIP, proREF6::REF6-HA/ref6-1 (N_WL and N_SH 8 h) and proREF6::REF6-HA/pif7-1 (N_SH 8 h) seedlings were prepared for each genotype of plants grown under white light and shade conditions (N, 4-day-old seedlings + 8 h white light + 40 h white light + 8 h triggering shade) using an anti-HA antibody (Lumiprobe sc-7392 X). The sample library was sequenced on a NovaSeq6000 PE150 by Shanghai Jiayin Biotechnology Ltd. A quality distribution plot and base content distribution were generated using FASTQC. Before read mapping, clean reads were obtained from the raw reads by removing adaptor sequences. The clean reads were aligned to the reference genome sequences using the BWA programme. The bam file was generated using the unique mapped reads as an input file, and using macs3 software for call peak with cutoff fold change > 2.0 and q < 0.05. The HOMER’s findMotifsGenome.pl tool was used for Motif analysis. The input file is the peak file and the genome fasta file. The DNA sequence is extracted according to the peak file, and the sequence is compared with the Motif database to obtain the Motif. DAVID was used to identify gene ontology enrichment in the ChIP–seq data. Visualization was performed with IGV version 2.7.2. Heatmaps and ChIP-seq profiles were generated using deepTools utilities plotHeatmap and plotProfile.

Firefly luciferase complementation imaging assays

The fragments encoding PIF7 were amplified by PCR and ligated into a pCAMBIA2300-nLUC vector to produce nLUC-PIF7. The coding regions of REF6C-terminal (3538-4083 bp) or ELF6C-terminal (3439-4023 bp) were amplified by PCR and ligated into pCAMBIA2300-cLUC to produce REF6C-cLUC or ELF6C-cLUC. The resulting constructs were transformed into Agrobacterium strain GV3101. Agrobacterium cells harbouring different constructs were then infiltrated into N. benthamiana leaves. Three days after infiltration, luciferin (Promega, USA) (2.5 mM, 0.1% Triton X-100) was spread before LUC activity was monitored by a Tanon 5500 chemical luminescence imaging system (Tanon, China).

Protein pull-down assay

The full-length PIF7 CDS was cloned and inserted into pCold-His-TF (Takara, Beijing, China). The plasmid was transformed into Escherichia coli strain Rosetta (DE3), and protein production was induced by incubation in 200 mL of LB medium containing 0.5 mM IPTG at 16 °C for 16 h. The His-TF-PIF7 protein was bound to Ni NTA Beads 6FF (Smart-Lifesciences, SA005025) by incubation for approximately 2 h at 4 °C. In the GST pull-down assay, the TF-His-PIF7 protein was incubated with pretreated GST-REF6C or GST-ELF6C beads for 2 h. GST was used as the negative control. The beads were resuspended in SDS‒PAGE loading buffer and analysed by SDS‒PAGE and immunoblotting using the anti-His antibody.

Co-IP assay

Total protein extracts were prepared from proREF6::REF6-HA/ref6-1 *PIF7-Flash seedlings grown under white light for 7 d and then treated with shade for 1 h or 8 h or maintained under white light. Anti-HA beads or anti-Flag beads were used to precipitate REF6-HA or PIF7-Flash from proREF6::REF6-HA/ref6-1*PIF7-Flash seedlings. PIF7-Flash and REF6-HA were detected by anti-Flag and anti-HA antibodies.

Statistics & reproducibility

All values are presented as means ± SEM or SD and differences for which P < 0.05 were considered significant. Significance levels were defined as n.s. (not significant, P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The number (n) of samples for each value is indicated in source data. Unless otherwise indicated, significant differences between the two samples were analysed with a two-tailed Student’s t-test as indicated in figure legends. Significant differences for multiple comparisons were determined by one-way ANOVA with Tukey’s HSD test as indicated in figure legends. Each experiment was repeated independently at least two times with consistent results.

Antibody information

The following antibodies were purchased: anti-Myc antibody (GNI, GNI4410-MC, 1:4000); anti-Flag antibody (GNI, GNI4410-FG, 1:1000); anti-β-Tubulin antibody (Abmart, M30109M, 1:4000); anti-GST antibody (Abmart, M20007, 1:4000); anti-His antibody (GNI, GNI4110-HS, 1:4000); anti-H3K27me3 antibody (Millipore 07-449, 1:1000); Anti-HA antibody (Lumiprobe sc-7392 X, 1:800); Glutathione Resin (GenScript, L00206); rProtein A Beads 4FF (Smart-Lifesciences, SA015005); Ni NTA Beads 6FF (Smart-Lifesciences, SA005025); Anti-DYKDDDDK Affinity Beads (Smart-Lifesciences, SA042100); Anti-HA Affinity Beads (Smart-Lifesciences, SA068100).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

RNA-seq and ChIP-seq data have been deposited in the NCBI GEO database with accession number GSE226745 and GSE268785. Source data are provided with this paper.

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Acknowledgements

We are grateful to Prof. Guodong Ren (Fundan University) for sharing ref6-1, ref6-3, elf6-3, ref6-1elf6-3, proREF6::REF6-HA/ref6-1 and clf28, Prof Jianxiang Liu (Zhejiang University) for sharing atx1-2, sdg25-1 and atx1-sdg25 mutants, and Prof. Xiaofeng Cao (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for sharing the seeds of jmj13g. This research was supported by funding provided by the National Natural Science Foundation of China to L.L. (NSFC32030018), and W.-H.S. received support from the Centre National de la Recherche Scientifique (Laboratoire International Associé Plant Epigenetic Research) and the Agence National de la Recherche (ANR-19-CE20-0018).

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State Key Laboratory of Genetic Engineering, Institute of Plants Biology, School of Life Sciences, Fudan University, Shanghai, People’s Republic of China
Qican Cheng, Yue Zeng, Sha Huang, Yu Xie & Lin Li
Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, School of Life Sciences, Fudan University, Shanghai, China
Chuanwei Yang
Institut de Biologie Moléculaire des Plantes, CNRS, Université de Strasbourg, Strasbourg, France
Wen-Hui Shen

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Qican Cheng
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Yue Zeng
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Sha Huang
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Chuanwei Yang
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Yu Xie
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Wen-Hui Shen
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Lin Li
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L.L. designed the experiments; Q.C. performed most of the experiments with the assistance from Y.Z., S.H., C.Y., and Y.X.; Q.C., L.L. and W.-H.S. analysed the data; L.L. wrote the manuscript and revised the manuscript with W.-H.S.

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Correspondence to Lin Li.

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Cheng, Q., Zeng, Y., Huang, S. et al. PHYTOCHROME-INTERACTING FACTOR 7 and RELATIVE OF EARLY FLOWERING 6 act in shade avoidance memory in Arabidopsis. Nat Commun 15, 8032 (2024). https://doi.org/10.1038/s41467-024-51834-4

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Received: 25 October 2023
Accepted: 08 August 2024
Published: 13 September 2024
DOI: https://doi.org/10.1038/s41467-024-51834-4

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Abstract

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Introduction

Results

Arabidopsis seedlings exhibit shade avoidance memory

PIF7 and REF6 are required for shade avoidance memory and the shade avoidance response

REF6 demethylates H3K27me3 on late shade-induced genes

Shade barely affects global genome-wide binding of REF6

REF6 and PIF7 regulate the expression of shade-memory-related genes

Demethylation of H3K27me3 at certain shade-memory-related genes is mediated by PIF7 and REF6

Enrichment of REF6 at certain shade-memory-related genes is dependent on PIF7

PIF7 binds REF6

Discussion

Methods

Plant materials and growth conditions

Hypocotyl measurement

RNA-seq analysis

Quantitative RT‒PCR analysis

ChIP‒qPCR assay

ChIP-seq analysis

Firefly luciferase complementation imaging assays

Protein pull-down assay

Co-IP assay

Statistics & reproducibility

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Reporting summary

Data availability

References

Acknowledgements

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Contributions

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Supplementary information

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