
==== Front
Nat Commun
Nat Commun
Nature Communications
2041-1723
Nature Publishing Group UK London

51834
10.1038/s41467-024-51834-4
Article
PHYTOCHROME-INTERACTING FACTOR 7 and RELATIVE OF EARLY FLOWERING 6 act in shade avoidance memory in Arabidopsis
Cheng Qican 1
Zeng Yue 1
Huang Sha 1
Yang Chuanwei 2
Xie Yu 1
Shen Wen-Hui 3
http://orcid.org/0000-0003-4840-5245
Li Lin linli@fudan.edu.cn

1
1 grid.8547.e 0000 0001 0125 2443 State Key Laboratory of Genetic Engineering, Institute of Plants Biology, School of Life Sciences, Fudan University, Shanghai, People’s Republic of China
2 https://ror.org/013q1eq08 grid.8547.e 0000 0001 0125 2443 Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, School of Life Sciences, Fudan University, Shanghai, China
3 grid.11843.3f 0000 0001 2157 9291 Institut de Biologie Moléculaire des Plantes, CNRS, Université de Strasbourg, Strasbourg, France
13 9 2024
13 9 2024
2024
15 803225 10 2023
8 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
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.

Cheng et al. find that priming shade treatment can be memorized and triggers faster hypocotyl elongation upon subsequent exposure to shade. During shade memory, PIF7 and REF6 are required to maintain low levels of H3K27me3 and hyper-induction of shade-memory-related genes.

Subject terms

Light responses
Epigenomics
Plant signalling
National Natural Science Foundation of China to L.L.(NSFC32030018)issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

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 herbivores2,4.

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 plants7,8. 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 shade8,9. 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 reversible10,11. 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 shade9,15. 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.

a New growth and growth rates and of 6-day-old Col-0 seedlings treated with shade (SH) white light (WL). New growth and real-time growth rate were recorded at 15-min intervals. Shade triggered growth rates of Col-0 seedlings grown under naïve (N) and 2 (b), 4 (c), 8 (d) or 10 (e) h shade-primed (P) conditions. The top panel is real-time growth rates recorded at 15-min intervals for 8 h captured by DynaPlant system. The bottom panel is average growth rates calculated from new growth in four-time spans: I (0–0.75 h), II (0.75–1.75 h), III (1.75–3 h), IV (3–8 h). Data are presented as mean values +/− SEM. Student’s two-sided t-test was used to calculate the P value between the indicated spans, and significant differences are shown by *(P < 0.05), ***(P < 0.001) and ns (P > 0.05). Source data are provided as a Source Data file.

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.

Shade-triggered growth rates and average growth rates of Col-0, ref6-1, proREF6::REF6-HA/ref6-1, ref6-3, elf6-3, ref6-1elf6-3 and pif7-1 seedlings grown under P (a) and N (b) conditions. The left panel shows real-time growth rates calculated from new growth which was captured by DynaPlant system. The right panel is average growth rates in four time spans. Data are presented as mean values +/− SEM. Student’s two-sided t-test was used to calculate the P value between the indicated genotypes, and significant differences are shown by **(P < 0.01), ***(P < 0.001) and ****(P < 0.0001). Source data are provided as a Source Data file.

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.

a Venn diagram showing the overlapping genes between 1 h and 7 h shade-induced genes and REF6 bound genes. The source data for shade-induced genes at 1 h and 7 h are from GSE59722. The source data for REF6 binding genes are from GSE65329. The P value was calculated by Fisher’s exact test. b Gene Ontology (GO) analysis of REF6-bound 7 h shade-induced genes. For each point, the size is proportional to the number of genes, and the colours represent the P value, Fisher’s one-tailed test. c Average density plot (left) and violin plot (right) showing the distribution profile of H3K27me3 levels on control genes (all non-induced genes by shade) and 7 h shade-induced genes. d Average density plot (left) and violin plot (right) showing the distribution profile of H3K27me3 levels of 7 h shade-induced genes in the Col-0 and ref6-1. e Average density plot (left) and violin plot (right) showing the distribution profile of H3K27me3 levels of REF6-bound 7 h shade-induced genes in Col-0 and ref6-1. f Relative expression levels of LNG2, EXPA11, XTH22 and XTH23 in Col-0 and ref6-1. g H3K27me3 enrichment on the LNG2, EXPA11, XTH22 and XTH23 loci in Col-0 and ref6-1. The top panels show a schematic representation of the gene structures and the positions of primers. The bottom panels show the effects of shade on H3K27me3 enrichment. The enrichment of H3K27me3 was calculated as input%. In c–e, the source data are from GSE65329. The P value was calculated by Welch’s t-test (two-sided). In f and g, seedlings that were grown under white light for 6 d and then maintained under white light or transferred to shade for 1 h and 8 h. Data are presented as mean values +/− SD (n = 3, where n refers to biological replicates). Different letters indicate statistically significant differences (P < 0.05) by one-way ANOVA with Tukey’s HSD test. Source data are provided as a Source Data file.

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 avoidance21–25. 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.

a Scatterplots of normalized REF6 ChIP-seq signal intensity in log2 for overlap regions between the N_WL and N_SH 8 h conditions. The profiles were generated after merging two biological replicates. The P value was calculated by t-test (two-sided). Average density plot (b) and violin plot (c) displaying the REF6-HA enrichement on N_WL&N_SH 8 h REF6 bound genes (5109) under N_WL and N_SH 8 h conditions. The P value was calculated by Welch’s t-test (two-sided). The profiles were generated after merging two biological replicates. d Representative genome browser view of REF6-HA binding to the LNG2, EXPA11, XTH22 and XTH23 loci in proREF6::REF6-HA/ref6-1 under N_WL and N_SH 8 h conditions. e ChIP‒qPCR analysis of REF6-HA enrichment using an anti-HA antibody at the LNG2, EXPA11, XTH22 and XTH23 loci in proREF6::REF6-HA/ref6-1. TA3 is a transposon and served as a negative control. The top panels show a schematic representation of the gene structures and positions of primers. REF6 enrichment was calculated as input%. Different letters indicate statistically significant differences (P < 0.05) based on one-way ANOVA with Tukey’s HSD test. Data are presented as mean values +/− SD (n = 3, where n refers to technical replicates). Source data are provided as a Source Data file.

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 datasets15,27. 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.

a Boxplot displaying the shade induction of shade-memory-related genes in Col-0, pif7-1 and ref6-1 under N and P conditions. The P value was calculated by the Mann–Whitney U test (two-sided). b Gene Ontology (GO) analysis of shade-memory-related genes. For each point, the size is proportional to the number of genes, and the colours represent the P value, Fisher’s one-tailed test. c Mfuzz clustering of differentially expressed shade-memory-related genes in Col-0, pif7-1 and ref6-1. Boxplots show the median (horizontal line), second to third quartiles (box), and whiskers extend to a maximum of 1.5× interquartile range beyond the box. GO analysis of genes in the C1 and C3 clusters are presented on the right side of heatmap. Orange and blue indicate RNA expression at high and low levels, respectively. d Relative expression levels of IAA19, BGL1 and GH3.6 in Col-0, pif7-1 and ref6-1 seedlings. The sampling conditions are shown in Supplementary Fig. 5a. Different letters indicate statistically significant differences (P < 0.05) based on one-way ANOVA with Tukey’s HSD test. Data are presented as mean values +/− SD (n = 3, where n refers to biological replicates). Source data are provided as a Source Data file.

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 avoidance7,29–34 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).Fig. 6 Demethylation of H3K27me3 at certain shade-memory-related genes is mediated by PIF7 and REF6.

a Violin plot displaying the REF6 enrichement under N_WL and N_SH 8 h conditions on shade-memory-related genes co-regulated by REF6 and PIF7. b Venn diagram showing the overlapping genes between shade-increased REF6 binding genes (N_SH 8 h vs. N_WL > 1.2), PIF7 bound genes and shade-memory-related genes co-regulated by REF6 and PIF7. The source data for PIF7 bound genes were obtained from GSE156584. c Gene Ontology (GO) analysis of PIF7 targets (bound and regulated genes), PIF7 and REF6 cotargets (bound and regulated genes) and REF6 targets (bound and regulated genes) in shade-memory-related genes. For each point, the size is proportional to the number of genes, and the colours represent the P value, Fisher’s one-tailed test. d Violin plot displaying the REF6 enrichement of REF6 and PIF7 co-targets under N_WL and N_SH 8 h. e ChIP‒qPCR analysis of PIF7 enrichment using an anti-MYC antibody at the IAA19, GH3.6 and BGL1 in 35S::PIF7-Flash. f ChIP‒qPCR analysis of H3K27me3 enrichment using anti-H3K27me3 antibody at the IAA19, GH3.6 and BGL1 loci in Col-0, pif7-1 and ref6-1. In a and d, the P value was calculated in a window from the 3 kb upstream REF6 binding summit to the 3 kb downstream by Welch’s t-test (two-sided). In e and f, the sampling conditions are shown in Supplementary Fig. 6a. The top panels show a schematic representation of the gene structures and position of primer and enrichment was calculated as input%. Different letters indicate statistically significant differences (P < 0.05) based on one-way ANOVA with Tukey’s HSD test. Data are presented as mean values +/− SD (n = 3, where n refers to biological replicates). Source data are provided as a Source Data file.

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.Fig. 7 Enrichment of REF6 at certain shade-memory-related genes is dependent on PIF7.

a Venn diagram showing the overlapping genes between N_SH 8 h REF6 bound genes and N_SH 8 h REF6 bound genes in proREF6::REF6-HA/pif7-1. The profiles were generated after merging two biological replicates. b Scatterplots of normalized REF6 ChIP-seq signal intensity in log2 scale of REF6 bound genes (4760) in proREF6::REF6-HA/ref6-1 and proREF6::REF6-HA/pif7-1 under N_SH 8 h conditions. The profiles were generated after merging two biological replicates. The P value was calculated by t-test (two-sided). c Average density plot (left) and violin plot (right) displaying the REF6-HA enrichment of REF6 bound genes (4760) in proREF6::REF6-HA/ref6-1 and proREF6::REF6-HA/pif7-1 under N_SH 8 h condition. The profiles were generated after merging two biological replicates. d Gene Ontology (GO) analysis of increased and decreased N_SH 8 h REF6 bound genes of proREF6::REF6-HA/pif7-1 vs. proREF6::REF6-HA/ref6-1 (FC > 1.2). For each point, the size is proportional to the number of genes, and the colours represent the P value, Fisher’s one-tailed test. e Venn diagram showing the overlapping genes between decreased REF6 bound genes in proREF6::REF6-HA/pif7-1, PIF7 bound genes and shade-memory-related genes co-regulated by REF6 and PIF7. f Violin plot displaying the REF6-HA enrichment of REF6 and PIF7 co-targets (17) in proREF6::REF6-HA/ref6-1 and proREF6::REF6-HA/pif7-1 under N_SH 8 h condition. g ChIP‒qPCR analysis of REF6-HA enrichment using an anti-HA antibody at the IAA19, BGL1, and GH3.6 loci in proREF6::REF6-HA/ref6-1 and proREF6::REF6-HA/pif7-1. The sampling conditions are shown in Supplementary Fig. 6a. The top panels show a IGV screenshots of REF6 binding peaks and the gene structure schematic of IAA19, BGL1 and GH3.6. REF6 enrichment was calculated as input%. Different letters indicate statistically significant differences (P < 0.05) based on one-way ANOVA with Tukey’s HSD test. Data are presented as mean values +/− SD (n = 3, where n refers to biological replicates). In c and f, the P value was calculated in a window from the 3 kb upstream REF6 binding summit to the 3 kb downstream by Welch’s t-test (two-sided).Source data are provided as a Source Data file.

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 reports8,37. 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.

a Interaction between PIF7 and the REF6C-terminal as detected by a luciferase complementation imaging (LCI) assay in Nicotiana benthamiana. b Interaction between PIF7 and the REF6C-terminal as detected by an in vitro GST pull-down assay. TF-His-fused PIF7 and GST-REF6C-terminal were purified from E. coli. c Interaction between PIF7 and REF6 in Arabidopsis as detected by an in vivo Co-IP assay. Anti-HA beads were used to precipitate REF6-HA from proREF6::REF6-HA/ref6-1*35S::PIF7-Flash seedlings grown under N_WL and N_SH 1 h. PIF7 and REF6 were detected by anti-Flag and anti-HA antibodies. d Shade effects on the interactions between PIF7 and REF6 in Arabidopsis was examined by Co-IP assays. Anti-Flag beads were used to precipitate PIF7-Flash from proREF6::REF6-HA/ref6-1*35S::PIF7-Flash seedlings grown under N_WL, N_SH 1 h and N_SH 8 h. PIF7 and REF6 were detected by anti-Flag and anti-HA antibodies. e Hypocotyl lengths of Col-0, pif7-1, ref6-1, ref6-3, pif7-1ref6-1 and pif7-1ref6-3 grown under P conditions which are shown in supplementary Fig. 1d. f Shade-triggered growth rates and average growth rates of Col-0, pif7-1ref6-1 and pif7-1ref6-3 seedlings under N and P conditions. g Proposed working model of the PIF7 and REF6 during shade-avoidance memory. Under white light, the chromatin of certain shade-memory-related genes carry repressive histone modifications (H3K27me3) that inhibit spontaneous transcription. After 8 h priming shade, the accumulated PIF7 recruits REF6 to demethylate H3K27me3 and activates these certain shade-memory-related genes (e.g., PRE1, YUCCA2, IAA19, GH3.6 and BGL1). During recovering white light condition, the expression levels of these certain shade-memory-related genes returned to normal levels in white light, but low levels of H3K27me3 were primed on these genes. When shade reoccurs, PIF7 quickly activates their expressions. In e and f, data are presented as mean values +/− SD (e) and SE (f) and the P value was calculated by One-way ANOVA with Tukey’s HSD test (e) and Student’s two-sided t-test (f), respectively, and significant differences are shown by ****(P < 0.0001). Source data are provided as a Source Data file.

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 memory38–41.

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 occupancy39,43. 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)48–50. 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.

Supplementary information

Supplementary Information

Peer Review File

Description Of Additional Supplementary File

Supplementary Data 1

Supplementary Data 2

Supplementary Data 3

Supplementary Data 4

Supplementary Data 5

Supplementary Data 6

Supplementary Data 7

Supplementary Data 8

Supplementary Data 9

Supplementary Data 10

Supplementary Data 11

Supplementary Data 12

Reporting summary

Source data

Source Data

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-024-51834-4.

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).

Author contributions

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.

Peer review

Peer review information

Nature Communications thanks Jorge Casal, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Casal JJ Shade avoidance Arabidopsis Book 2012 10 e0157 10.1199/tab.0157 22582029
Casal, J. J. Shade avoidance. Arabidopsis Book 10, e0157 (2012).22582029 10.1199/tab.0157
2. Ballare CL Pierik R The shade-avoidance syndrome: multiple signals and ecological consequences Plant Cell Environ. 2017 40 2530 2543 10.1111/pce.12914 28102548
Ballare, C. L. & Pierik, R. The shade-avoidance syndrome: multiple signals and ecological consequences. Plant Cell Environ. 40, 2530–2543 (2017).28102548 10.1111/pce.12914
3. Cerdan PD Chory J Regulation of flowering time by light quality Nature 2003 423 881 885 10.1038/nature01636 12815435
Cerdan, P. D. & Chory, J. Regulation of flowering time by light quality. Nature 423, 881–885 (2003).12815435 10.1038/nature01636
4. Izaguirre MM Mazza CA Biondini M Baldwin IT Ballare CL Remote sensing of future competitors: impacts on plant defenses Proc. Natl. Acad. Sci. USA 2006 103 7170 7174 10.1073/pnas.0509805103 16632610
Izaguirre, M. M., Mazza, C. A., Biondini, M., Baldwin, I. T. & Ballare, C. L. Remote sensing of future competitors: impacts on plant defenses. Proc. Natl. Acad. Sci. USA 103, 7170–7174 (2006).16632610 10.1073/pnas.0509805103
5. Schmitt J Dudley SA Pigliucci M Manipulative approaches to testing adaptive plasticity: phytochrome-mediated shade-avoidance responses in plants Am. Nat. 1999 154 S43 S54 10.1086/303282 29586708
Schmitt, J., Dudley, S. A. & Pigliucci, M. Manipulative approaches to testing adaptive plasticity: phytochrome-mediated shade-avoidance responses in plants. Am. Nat. 154, S43–S54 (1999).29586708 10.1086/303282
6. Leivar P Quail PH PIFs: pivotal components in a cellular signaling hub Trends Plant Sci. 2011 16 19 28 10.1016/j.tplants.2010.08.003 20833098
Leivar, P. & Quail, P. H. PIFs: pivotal components in a cellular signaling hub. Trends Plant Sci. 16, 19–28 (2011).20833098 10.1016/j.tplants.2010.08.003
7. Burko Y PIF7 is a master regulator of thermomorphogenesis in shade Nat. Commun. 2022 13 4942 10.1038/s41467-022-32585-6 36038577
Burko, Y. et al. PIF7 is a master regulator of thermomorphogenesis in shade. Nat. Commun. 13, 4942 (2022).36038577 10.1038/s41467-022-32585-6
8. Li L Linking photoreceptor excitation to changes in plant architecture Genes. Dev. 2012 26 785 790 10.1101/gad.187849.112 22508725
Li, L. et al. Linking photoreceptor excitation to changes in plant architecture. Genes. Dev. 26, 785–790 (2012).22508725 10.1101/gad.187849.112
9. Willige BC PHYTOCHROME-INTERACTING FACTORs trigger environmentally responsive chromatin dynamics in plants Nat. Genet. 2021 53 955 961 10.1038/s41588-021-00882-3 34140685
Willige, B. C. et al. PHYTOCHROME-INTERACTING FACTORs trigger environmentally responsive chromatin dynamics in plants. Nat. Genet. 53, 955–961 (2021).34140685 10.1038/s41588-021-00882-3
10. Cole B Kay SA Chory J Automated analysis of hypocotyl growth dynamics during shade avoidance in Arabidopsis Plant J. 2011 65 991 1000 10.1111/j.1365-313X.2010.04476.x 21288269
Cole, B., Kay, S. A. & Chory, J. Automated analysis of hypocotyl growth dynamics during shade avoidance in Arabidopsis. Plant J. 65, 991–1000 (2011).21288269 10.1111/j.1365-313X.2010.04476.x
11. Hwang YS Quail PH Phytochrome-regulated PIL1 derepression is developmentally modulated Plant Cell Physiol. 2008 49 501 511 10.1093/pcp/pcn024 18296722
Hwang, Y. S. & Quail, P. H. Phytochrome-regulated PIL1 derepression is developmentally modulated. Plant Cell Physiol. 49, 501–511 (2008).18296722 10.1093/pcp/pcn024
12. Salter MG Franklin KA Whitelam GC Gating of the rapid shade-avoidance response by the circadian clock in plants Nature 2003 426 680 683 10.1038/nature02174 14668869
Salter, M. G., Franklin, K. A. & Whitelam, G. C. Gating of the rapid shade-avoidance response by the circadian clock in plants. Nature 426, 680–683 (2003).14668869 10.1038/nature02174
13. Peng M Linking PHYTOCHROME-INTERACTING FACTOR to histone modification in plant shade avoidance Plant Physiol. 2018 176 1341 1351 10.1104/pp.17.01189 29187567
Peng, M. et al. Linking PHYTOCHROME-INTERACTING FACTOR to histone modification in plant shade avoidance. Plant Physiol. 176, 1341–1351 (2018).29187567 10.1104/pp.17.01189
14. Nguyen NH HISTONE DEACETYLASE 9 promotes hypocotyl-specific auxin response under shade Plant J. 2023 10.1111/tpj.16410 37609706
Nguyen, N. H. et al. HISTONE DEACETYLASE 9 promotes hypocotyl-specific auxin response under shade. Plant J.10.1111/tpj.16410 (2023).37609706 10.1111/tpj.16410
15. Yang C PIF7-mediated epigenetic reprogramming promotes the transcriptional response to shade in Arabidopsis EMBO J. 2023 42 e111472 10.15252/embj.2022111472 36912149
Yang, C. et al. PIF7-mediated epigenetic reprogramming promotes the transcriptional response to shade in Arabidopsis. EMBO J. 42, e111472 (2023).36912149 10.15252/embj.2022111472
16. Zhu T Yang C Xie Y Huang S Li L Shade-induced lncRNA PUAR promotes shade response by repressing PHYA expression EMBO Rep. 2023 24 e56105 10.15252/embr.202256105 36970931
Zhu, T., Yang, C., Xie, Y., Huang, S. & Li, L. Shade-induced lncRNA PUAR promotes shade response by repressing PHYA expression. EMBO Rep. 24, e56105 (2023).36970931 10.15252/embr.202256105
17. Harris CJ Amtmann A Ton J Epigenetic processes in plant stress priming: open questions and new approaches Curr. Opin. Plant Biol. 2023 75 102432 10.1016/j.pbi.2023.102432 37523900
Harris, C. J., Amtmann, A. & Ton, J. Epigenetic processes in plant stress priming: open questions and new approaches. Curr. Opin. Plant Biol. 75, 102432 (2023).37523900 10.1016/j.pbi.2023.102432
18. Huang S Yang C Li L Unraveling the dynamic integration of auxin, brassinosteroid and gibberellin in early shade-induced hypocotyl elongation Phenomics 2022 2 119 129 10.1007/s43657-022-00044-3 36939748
Huang, S., Yang, C. & Li, L. Unraveling the dynamic integration of auxin, brassinosteroid and gibberellin in early shade-induced hypocotyl elongation. Phenomics 2, 119–129 (2022).36939748 10.1007/s43657-022-00044-3
19. Cui X REF6 recognizes a specific DNA sequence to demethylate H3K27me3 and regulate organ boundary formation in Arabidopsis Nat. Genet. 2016 48 694 699 10.1038/ng.3556 27111035
Cui, X. et al. REF6 recognizes a specific DNA sequence to demethylate H3K27me3 and regulate organ boundary formation in Arabidopsis. Nat. Genet. 48, 694–699 (2016).27111035 10.1038/ng.3556
20. Huang S Shade-induced RTFL/DVL peptides negatively regulate the shade response by directly interacting with BSKs in Arabidopsis Nat. Commun. 2023 14 6898 10.1038/s41467-023-42618-3 37898648
Huang, S. et al. Shade-induced RTFL/DVL peptides negatively regulate the shade response by directly interacting with BSKs in Arabidopsis. Nat. Commun. 14, 6898 (2023).37898648 10.1038/s41467-023-42618-3
21. Hwang G PIF4 promotes expression of LNG1 and LNG2 to induce thermomorphogenic growth in Arabidopsis Front Plant Sci. 2017 8 1320 10.3389/fpls.2017.01320 28791042
Hwang, G. et al. PIF4 promotes expression of LNG1 and LNG2 to induce thermomorphogenic growth in Arabidopsis. Front Plant Sci. 8, 1320 (2017).28791042 10.3389/fpls.2017.01320
22. Song B Phytochrome A inhibits shade avoidance responses under strong shade through repressing the brassinosteroid pathway in Arabidopsis Plant J. 2020 104 1520 1534 10.1111/tpj.15018 33037720
Song, B. et al. Phytochrome A inhibits shade avoidance responses under strong shade through repressing the brassinosteroid pathway in Arabidopsis. Plant J. 104, 1520–1534 (2020).33037720 10.1111/tpj.15018
23. Guo H Three related receptor-like kinases are required for optimal cell elongation in Arabidopsis thaliana Proc. Natl. Acad. Sci. USA 2009 106 7648 7653 10.1073/pnas.0812346106 19383785
Guo, H. et al. Three related receptor-like kinases are required for optimal cell elongation in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 106, 7648–7653 (2009).19383785 10.1073/pnas.0812346106
24. Claisse G Charrier B Kreis M The Arabidopsis thaliana GSK3/Shaggy like kinase AtSK3-2 modulates floral cell expansion Plant Mol. Biol. 2007 64 113 124 10.1007/s11103-007-9138-y 17427040
Claisse, G., Charrier, B. & Kreis, M. The Arabidopsis thaliana GSK3/Shaggy like kinase AtSK3-2 modulates floral cell expansion. Plant Mol. Biol. 64, 113–124 (2007).17427040 10.1007/s11103-007-9138-y
25. Polko JK Ethylene-induced differential petiole growth in Arabidopsis thaliana involves local microtubule reorientation and cell expansion N. Phytol. 2012 193 339 348 10.1111/j.1469-8137.2011.03920.x
Polko, J. K. et al. Ethylene-induced differential petiole growth in Arabidopsis thaliana involves local microtubule reorientation and cell expansion. N. Phytol. 193, 339–348 (2012).10.1111/j.1469-8137.2011.03920.x
26. Qiu Q DNA methylation repels targeting of Arabidopsis REF6 Nat. Commun. 2019 10 2063 10.1038/s41467-019-10026-1 31048693
Qiu, Q. et al. DNA methylation repels targeting of Arabidopsis REF6. Nat. Commun. 10, 2063 (2019).31048693 10.1038/s41467-019-10026-1
27. Kohnen MV Neighbor detection induces organ-specific transcriptomes, revealing patterns underlying hypocotyl-specific growth Plant Cell 2016 28 2889 2904 10.1105/tpc.16.00463 27923878
Kohnen, M. V. et al. Neighbor detection induces organ-specific transcriptomes, revealing patterns underlying hypocotyl-specific growth. Plant Cell 28, 2889–2904 (2016).27923878 10.1105/tpc.16.00463
28. D’Urso A Brickner JH Mechanisms of epigenetic memory Trends Genet. 2014 30 230 236 10.1016/j.tig.2014.04.004 24780085
D’Urso, A. & Brickner, J. H. Mechanisms of epigenetic memory. Trends Genet. 30, 230–236 (2014).24780085 10.1016/j.tig.2014.04.004
29. Zhou XY Song L Xue HW Brassinosteroids regulate the differential growth of Arabidopsis hypocotyls through auxin signaling components IAA19 and ARF7 Mol. Plant 2013 6 887 904 10.1093/mp/sss123 23125315
Zhou, X. Y., Song, L. & Xue, H. W. Brassinosteroids regulate the differential growth of Arabidopsis hypocotyls through auxin signaling components IAA19 and ARF7. Mol. Plant 6, 887–904 (2013).23125315 10.1093/mp/sss123
30. Nakazawa M DFL1, an auxin-responsive GH3 gene homologue, negatively regulates shoot cell elongation and lateral root formation, and positively regulates the light response of hypocotyl length Plant J. 2001 25 213 221 11169197
Nakazawa, M. et al. DFL1, an auxin-responsive GH3 gene homologue, negatively regulates shoot cell elongation and lateral root formation, and positively regulates the light response of hypocotyl length. Plant J. 25, 213–221 (2001).11169197
31. Ikeda M Fujiwara S Mitsuda N Ohme-Takagi M A triantagonistic basic helix-loop-helix system regulates cell elongation in Arabidopsis Plant Cell 2012 24 4483 4497 10.1105/tpc.112.105023 23161888
Ikeda, M., Fujiwara, S., Mitsuda, N. & Ohme-Takagi, M. A triantagonistic basic helix-loop-helix system regulates cell elongation in Arabidopsis. Plant Cell 24, 4483–4497 (2012).23161888 10.1105/tpc.112.105023
32. Nozue K Shade avoidance components and pathways in adult plants revealed by phenotypic profiling PLoS Genet. 2015 11 e1004953 10.1371/journal.pgen.1004953 25874869
Nozue, K. et al. Shade avoidance components and pathways in adult plants revealed by phenotypic profiling. PLoS Genet. 11, e1004953 (2015).25874869 10.1371/journal.pgen.1004953
33. Lee S Overexpression of PRE1 and its homologous genes activates Gibberellin-dependent responses in Arabidopsis thaliana Plant Cell Physiol. 2006 47 591 600 10.1093/pcp/pcj026 16527868
Lee, S. et al. Overexpression of PRE1 and its homologous genes activates Gibberellin-dependent responses in Arabidopsis thaliana. Plant Cell Physiol. 47, 591–600 (2006).16527868 10.1093/pcp/pcj026
34. Staswick PE Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid Plant Cell 2005 17 616 627 10.1105/tpc.104.026690 15659623
Staswick, P. E. et al. Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell 17, 616–627 (2005).15659623 10.1105/tpc.104.026690
35. Yang C Two bHLH transcription factors, bHLH48 and bHLH60, associate with phytochrome interacting factor 7 to regulate hypocotyl elongation in Arabidopsis Cell Rep. 2021 35 109054 10.1016/j.celrep.2021.109054 33951433
Yang, C. et al. Two bHLH transcription factors, bHLH48 and bHLH60, associate with phytochrome interacting factor 7 to regulate hypocotyl elongation in Arabidopsis. Cell Rep. 35, 109054 (2021).33951433 10.1016/j.celrep.2021.109054
36. He K The histone H3K27 demethylase REF6/JMJ12 promotes thermomorphogenesis in Arabidopsis Natl. Sci. Rev. 2022 9 nwab213 10.1093/nsr/nwab213 35591912
He, K. et al. The histone H3K27 demethylase REF6/JMJ12 promotes thermomorphogenesis in Arabidopsis. Natl. Sci. Rev. 9, nwab213 (2022).35591912 10.1093/nsr/nwab213
37. Huang, X. et al. Shade-induced nuclear localization of PIF7 is regulated by phosphorylation and 14-3-3 proteins in Arabidopsis. Elife 7, 10.7554/eLife.31636 (2018).
38. Zhao, Z. et al. Sustained TNF-alpha stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness. Elife 9, 10.7554/eLife.61965 (2020).
39. Oberkofler V Pratx L Baurle I Epigenetic regulation of abiotic stress memory: maintaining the good things while they last Curr. Opin. Plant Biol. 2021 61 102007 10.1016/j.pbi.2021.102007 33571730
Oberkofler, V., Pratx, L. & Baurle, I. Epigenetic regulation of abiotic stress memory: maintaining the good things while they last. Curr. Opin. Plant Biol. 61, 102007 (2021).33571730 10.1016/j.pbi.2021.102007
40. Lamke J Baurle I Epigenetic and chromatin-based mechanisms in environmental stress adaptation and stress memory in plants Genome Biol. 2017 18 124 10.1186/s13059-017-1263-6 28655328
Lamke, J. & Baurle, I. Epigenetic and chromatin-based mechanisms in environmental stress adaptation and stress memory in plants. Genome Biol. 18, 124 (2017).28655328 10.1186/s13059-017-1263-6
41. Conrath U Beckers GJ Langenbach CJ Jaskiewicz MR Priming for enhanced defense Annu. Rev. Phytopathol. 2015 53 97 119 10.1146/annurev-phyto-080614-120132 26070330
Conrath, U., Beckers, G. J., Langenbach, C. J. & Jaskiewicz, M. R. Priming for enhanced defense. Annu. Rev. Phytopathol. 53, 97–119 (2015).26070330 10.1146/annurev-phyto-080614-120132
42. Hugues A Jacobs CS Roudier F Mitotic inheritance of PRC2-mediated silencing: mechanistic insights and developmental perspectives Front. Plant Sci. 2020 11 262 10.3389/fpls.2020.00262 32211012
Hugues, A., Jacobs, C. S. & Roudier, F. Mitotic inheritance of PRC2-mediated silencing: mechanistic insights and developmental perspectives. Front. Plant Sci. 11, 262 (2020).32211012 10.3389/fpls.2020.00262
43. Baurle I Can’t remember to forget you: chromatin-based priming of somatic stress responses Semin. Cell Dev. Biol. 2018 83 133 139 10.1016/j.semcdb.2017.09.032 28951121
Baurle, I. Can’t remember to forget you: chromatin-based priming of somatic stress responses. Semin. Cell Dev. Biol. 83, 133–139 (2018).28951121 10.1016/j.semcdb.2017.09.032
44. Avramova Z Transcriptional ‘memory’ of a stress: transient chromatin and memory (epigenetic) marks at stress-response genes Plant J. Cell Mol. Biol. 2015 83 149 159 10.1111/tpj.12832
Avramova, Z. Transcriptional ‘memory’ of a stress: transient chromatin and memory (epigenetic) marks at stress-response genes. Plant J. Cell Mol. Biol. 83, 149–159 (2015).10.1111/tpj.12832
45. Yamaguchi N H3K27me3 demethylases alter HSP22 and HSP17.6C expression in response to recurring heat in Arabidopsis Nat. Commun. 2021 12 3480 10.1038/s41467-021-23766-w 34108473
Yamaguchi, N. et al. H3K27me3 demethylases alter HSP22 and HSP17.6C expression in response to recurring heat in Arabidopsis. Nat. Commun. 12, 3480 (2021).34108473 10.1038/s41467-021-23766-w
46. Zhang J Highly enriched BEND3 prevents the premature activation of bivalent genes during differentiation Science 2022 375 1053 1058 10.1126/science.abm0730 35143257
Zhang, J. et al. Highly enriched BEND3 prevents the premature activation of bivalent genes during differentiation. Science 375, 1053–1058 (2022).35143257 10.1126/science.abm0730
47. Casal JJ Smith H Persistent effects of changes in phytochrome status on internode growth in light-grown mustard: Occurrence, kinetics and locus of perception Planta 1988 175 214 220 10.1007/BF00392430 24221715
Casal, J. J. & Smith, H. Persistent effects of changes in phytochrome status on internode growth in light-grown mustard: Occurrence, kinetics and locus of perception. Planta 175, 214–220 (1988).24221715 10.1007/BF00392430
48. Lu F Cui X Zhang S Jenuwein T Cao X Arabidopsis REF6 is a histone H3 lysine 27 demethylase Nat. Genet. 2011 43 715 719 10.1038/ng.854 21642989
Lu, F., Cui, X., Zhang, S., Jenuwein, T. & Cao, X. Arabidopsis REF6 is a histone H3 lysine 27 demethylase. Nat. Genet. 43, 715–719 (2011).21642989 10.1038/ng.854
49. Yan W Dynamic and spatial restriction of Polycomb activity by plant histone demethylases Nat. Plants 2018 4 681 689 10.1038/s41477-018-0219-5 30104650
Yan, W. et al. Dynamic and spatial restriction of Polycomb activity by plant histone demethylases. Nat. Plants 4, 681–689 (2018).30104650 10.1038/s41477-018-0219-5
50. Zheng S The Arabidopsis H3K27me3 demethylase JUMONJI 13 is a temperature and photoperiod dependent flowering repressor Nat. Commun. 2019 10 1303 10.1038/s41467-019-09310-x 30899015
Zheng, S. et al. The Arabidopsis H3K27me3 demethylase JUMONJI 13 is a temperature and photoperiod dependent flowering repressor. Nat. Commun. 10, 1303 (2019).30899015 10.1038/s41467-019-09310-x
51. Yu X Modulation of brassinosteroid-regulated gene expression by Jumonji domain-containing proteins ELF6 and REF6 in Arabidopsis Proc. Natl. Acad. Sci. USA 2008 105 7618 7623 10.1073/pnas.0802254105 18467490
Yu, X. et al. Modulation of brassinosteroid-regulated gene expression by Jumonji domain-containing proteins ELF6 and REF6 in Arabidopsis. Proc. Natl. Acad. Sci. USA 105, 7618–7623 (2008).18467490 10.1073/pnas.0802254105
52. Antunez-Sanchez, J. et al. A new role for histone demethylases in the maintenance of plant genome integrity. Elife 9, 10.7554/eLife.58533 (2020).
53. Crisp PA Ganguly D Eichten SR Borevitz JO Pogson BJ Reconsidering plant memory: Intersections between stress recovery, RNA turnover, and epigenetics Sci. Adv. 2016 2 e1501340 10.1126/sciadv.1501340 26989783
Crisp, P. A., Ganguly, D., Eichten, S. R., Borevitz, J. O. & Pogson, B. J. Reconsidering plant memory: Intersections between stress recovery, RNA turnover, and epigenetics. Sci. Adv. 2, e1501340 (2016).26989783 10.1126/sciadv.1501340
54. Liu J An H3K27me3 demethylase-HSFA2 regulatory loop orchestrates transgenerational thermomemory in Arabidopsis Cell Res. 2019 29 379 390 10.1038/s41422-019-0145-8 30778176
Liu, J. et al. An H3K27me3 demethylase-HSFA2 regulatory loop orchestrates transgenerational thermomemory in Arabidopsis. Cell Res. 29, 379–390 (2019).30778176 10.1038/s41422-019-0145-8
55. Wang X REF6 promotes lateral root formation through de-repression of PIN1/3/7 genes J. Integr. Plant Biol. 2019 61 383 387 10.1111/jipb.12726 30267471
Wang, X. et al. REF6 promotes lateral root formation through de-repression of PIN1/3/7 genes. J. Integr. Plant Biol. 61, 383–387 (2019).30267471 10.1111/jipb.12726
56. Song ZT Zhang LL Han JJ Zhou M Liu JX Histone H3K4 methyltransferases SDG25 and ATX1 maintain heat-stress gene expression during recovery in Arabidopsis Plant J. Cell Mol. Biol. 2021 105 1326 1338 10.1111/tpj.15114
Song, Z. T., Zhang, L. L., Han, J. J., Zhou, M. & Liu, J. X. Histone H3K4 methyltransferases SDG25 and ATX1 maintain heat-stress gene expression during recovery in Arabidopsis. Plant J. Cell Mol. Biol. 105, 1326–1338 (2021).10.1111/tpj.15114
