Precise regulation of flowering time in plants is achieved through tight transcriptional control of master regulators, which is fine-tuned by dynamic histone modifications and changes in chromatin structure. In our search for flowering-related regulatory networks, we identified METHYL-BINDING DOMAIN PROTEIN 4 (MBD4) as a key hub controlling flowering and responses to high ambient temperature in Brassica napus and Arabidopsis. While MBD4 was previously described as a DNA methylation reader, our findings reveal that MBD4 recognizes histone arginine methylation. The MBD4 genome-wide distribution and its coordinated expression with histone methylation writers coincide with global patterns of arginine methylation. Although histone lysine methylation is well understood, the role of arginine methylation remains largely unexplored. Our results demonstrate that MBD4 plays a critical role in the transcriptional control of flowering regulators by recognizing arginine methylation and facilitating acetylation removal. Additionally, we confirmed the significance of the Brassica napus MBD4 homeolog in modulating the transition to the reproductive phase through chromatin alterations at the BnaFLC and BnaMAF4 loci, suggesting that chromatin readers may have a global influence on important developmental traits in crops. Overall, understanding these molecular mechanisms is highly relevant to developing strategies to improve crop adaptation to global warming.

Chromatin remodeling plays a central role in regulating plant development and physiology by shaping the gene expression patterns that drive biological processes. Among epigenetic modifications, histone acetylation is particularly relevant as it alters chromatin structure and influences transcriptional activity. MYST-type histone acetyltransferases (HAT) are evolutionarily conserved components of the Nucleosome Acetyltransferase of histone H4 (NuA4) complex, a key regulator that acetylates histones H4, H2A, and the histone variant H2A.Z. Growing evidence supports the presence of a canonical NuA4-C in plants, similar to that described in yeast. In this review, we summarize recent studies that have begun to uncover its broad role in plant biology, highlighting its involvement in diverse processes such as the skoto- to photomorphogenesis switch, chloroplast development, shade avoidance responses, thermomorphogenesis, the vegetative-to-reproductive transition, plant growth, reproduction and hormonal signalling. In addition, we discuss recent advances in understanding the crosstalk of NuA4-C-mediated H4ac and H2A.Z deposition with other chromatin remodeling complexes in plants. Although significant progress has been made, a full understanding of the complex functions remains unavailable. Current evidence indicates that NuA4-C in yeast and TIP60 in humans are central regulators of transcription, acting not only through histone acetylation but also by influencing transcription elongation and RNA splicing, although direct evidence for similar functions in plant NuA4-C still remains limited. This regulatory role might be critical for integrating developmental programs with environmental signalling pathways. While initial insights into the recruitment of NuA4-C to target genes have emerged, further research is needed to clarify how its activity is controlled and modulated in different biological contexts.

Abstract Chromatin remodeling plays a central role in regulating plant development and physiology by shaping the gene expression patterns that drive biological processes. Among epigenetic modifications, histone acetylation is particularly relevant as it alters chromatin structure and influences transcriptional activity. MYST-type histone acetyltransferases (HAT) are evolutionarily conserved components of the Nucleosome Acetyltransferase of Histone H4 complex (NuA4-C), a key regulator that acetylates histones H4, H2A, and the histone variant H2A.Z. Growing evidence supports the presence of a canonical NuA4-C in plants, similar to that described in yeast. In this review, we summarize recent studies that have begun to uncover its broad role in plant biology, highlighting its involvement in diverse processes such as the skoto- to photomorphogenesis switch, chloroplast development, shade avoidance responses, thermomorphogenesis, the vegetative-to-reproductive transition, plant growth, reproduction, and hormonal signaling. In addition, we discuss recent advances in understanding the crosstalk of NuA4-C-mediated H4 acetylation and H2A.Z deposition with other chromatin remodeling complexes in plants. Although significant progress has been made, a full understanding of the complex functions remains unavailable. Current evidence indicates that NuA4-C in yeast and TIP60 in humans are central regulators of transcription, acting not only through histone acetylation but also by influencing transcription elongation and RNA splicing, although direct evidence for similar functions in plant NuA4-C still remains limited. This regulatory role might be critical for integrating developmental programs with environmental signaling pathways. While initial insights into the recruitment of NuA4-C to target genes have emerged, further research is needed to clarify how its activity is controlled and modulated in different biological contexts.

INHIBITOR OF GROWTH (ING) proteins are chromatin readers that bind trimethylated histone H3 lysine (K) 4 (H3K4me3) marks and associate with either histone acetyltransferase or deacetylase complexes to activate or repress gene transcription, respectively. In plants, there are two types of ING proteins, namely ING1 and ING2. Here, we report that Arabidopsis thaliana ING2 associates with multiple subunits of the histone H4 acetyltransferase complex NuA4, controls genome-wide levels of histone H4 acetylation (H4ac), and regulates different developmental processes, including the initiation of flowering. Our data indicate that ING2 biological functions are largely independent of ING1 activity. We find that ING2 is recruited to the chromatin of key floral integrators, such as FT and SOC1, and is required for their timely activation by modulating histone H4ac levels at these loci. Our observations reveal a genetic interaction between ING2 and genes encoding relevant proteins for H3K4me3 or H2A.Z deposition, suggesting that ING2 might represent a hub for potential crosstalk between histone H4ac and these histone modifications/variants.

Pyropia yezoensis, a key economic marine crop in Asia, serves as a model for red seaweed research. Previously, we determined that histone acetylation plays essential roles in the response to abiotic stresses and the formation of asexual spores in P. yezoensis. However, the reader proteins of acetylated histones remain to be identified. In this study, we characterized the gene encoding a YEATS domain-containing protein (PyYEATS) in P. yezoensis. The PyYEATS protein, mainly localized in nuclei with a small amount in cytosolic fractions in thalli, harbored a classic YEATS domain followed by a coiled-coil domain at the C-terminal. This locus exhibited conservation in domain structure compared with its homologs in other red algae and showed a closer relationship to animal GAS41 counterparts than plant YAFs. In vitro assays showed that the PyYEATS protein preferentially binds H3K14ac, H3K18ac, and H4K5ac and that the F105, W124, and F127 residues in the YEATS domain are essential for its affinity to these histone acetylation sites. Y2H and GST-pulldown approaches revealed an interaction of PyYEATS with S-adenosyl-L-homocysteine hydrolase (SAHase), eukaryotic translation initiation factor 3 (eIF3), and actin, among others, suggesting possible extra-transcriptional functions for PyYEATS. Finally, we observed that the transcript levels of this gene significantly increased after wounding in P. yezoensis thalli, pointing out a potential role in the P. yezoensis stress response. Our findings provide important insights into the evolution of chromatin readers of histone acetylation in red seaweeds and help to shed light on the biological function exerted by PyYEATS in this species.

INHIBITOR OF GROWTH (ING) proteins are chromatin readers that bind trimethylated histone H3 lysine (K) 4 (H3K4me3) marks and associate with either histone acetyltransferase or deacetylase complexes to activate or repress gene transcription, respectively. In plants, there are two types of ING proteins named ING1 and ING2. Here we report that Arabidopsis ING2 associates with multiple subunits of the histone H4 acetyltransferase complex NuA4, controls genome-wide levels of histone H4 acetylation (H4ac) and regulates different developmental processes including the initiation of flowering. Our data indicates that ING2 biological functions are largely independent of ING1 activity. We find that ING2 is recruited to the chromatin of key floral integrators such as FT and SOC1, and is required for their timely activation by modulating histone H4ac levels at these loci. Besides, our observations reveal a genetic interaction between ING2 and genes encoding relevant proteins for H3K4me3 or H2A.Z deposition, suggesting that ING2 might represent a hub for potential crosstalk between histone H4ac and these histone modifications/variants. ### Competing Interest Statement The authors have declared no competing interest.

Agriculture faces great challenges to overcome global warming and improve system sustainability, requiring access to novel genetic diversity. So far, wild populations and local landraces remain poorly explored. This is notably the case for the two diploid species, Brassica oleracea L. (CC, 2n=2x=18) and B. rapa L. (AA, 2n=2x=20). In order to explore the genetic diversity in both species, we have collected populations in their centre of origin, the Mediterranean basin, on a large contrasting climatic and soil gradient from northern Europe to southern sub-Saharan regions. In these areas, we also collected 14 populations belonging to five B. oleracea closely related species. Our objective was to ensure the absence of species misidentification at the seedling stage among the populations collected and to describe thereafter their origins. We combined flow cytometry, sequencing of a species-specific chloroplast genomic region, as well as cytogenetic analyses in case of unexpected results for taxonomic verification. Out of the 112 B. oleracea and 154 B. rapa populations collected, 103 and 146, respectively, presented a good germination rate and eighteen populations were misidentified. The most frequent mistake was the confusion of these diploid species with B. napus. Additionally for B. rapa, two autotetraploid populations were observed. Habitats of the collected and confirmed wild populations and landraces are described in this study. The unique plant material described here will serve to investigate the genomic regions involved in adaptation to climate and microbiota within the framework of the H2020 Prima project ‘BrasExplor’.

In Brassica rapa, the epigenetic modifier BraA.CLF orchestrates flowering by modulating H3K27me3 levels at the floral integrator genes FT, SOC1, and SEP3, thereby influencing their expression. CURLY LEAF (CLF) is the catalytic subunit of the plant Polycomb Repressive Complex 2 that mediates the trimethylation of histone H3 lysine 27 (H3K27me3), an epigenetic modification that leads to gene silencing. While the function of CURLY LEAF (CLF) has been extensively studied in Arabidopsis thaliana, its role in Brassica crops is barely known. In this study, we focused on the Brassica rapa homolog of CLF and found that the loss-of-function mutant braA.clf-1 exhibits an accelerated flowering together with pleiotropic phenotypic alterations compared to wild-type plants. In addition, we carried out transcriptomic and H3K27me3 genome-wide analyses to identify the genes regulated by BraA.CLF. Interestingly, we observed that several floral regulatory genes, including the B. rapa homologs of FT, SOC1 and SEP3, show reduced H3K27me3 levels and increased transcript levels compared to wild-type plants, suggesting that they are direct targets of BraA.CLF and key players in regulating flowering time in this crop. In addition, the results obtained will enhance our understanding of the epigenetic mechanisms regulating key developmental traits and will aid to increase crop yield by engineering new Brassica varieties with different flowering time requirements.

Elevated growth temperatures are negatively affecting crop productivity by increasing yield losses. The modulation of root traits associated with improved response to rising temperatures is a promising approach to generate new varieties better suited to face the environmental constraints caused by climate change. In this study, we identified several Brassica napus root traits altered in response to warm ambient temperatures. Different combinations of changes in specific root traits result in an extended and deeper root system. This overall root growth expansion facilitates root response by maximizing root–soil surface interaction and increasing roots’ ability to explore extended soil areas. We associated these traits with coordinated cellular events, including changes in cell division and elongation rates that drive root growth increases triggered by warm temperatures. Comparative transcriptomic analysis revealed the main genetic determinants of these root system architecture (RSA) changes and uncovered the necessity of a tight regulation of the heat-shock stress response to adjusting root growth to warm temperatures. Our work provides a phenotypic, cellular, and genetic framework of root response to warming temperatures that will help to harness root response mechanisms for crop yield improvement under the future climatic scenario.

Agriculture faces great challenges to overcome global warming and to improve system sustainability, requiring access to novel genetic diversity. So far, wild populations and local landraces remain poorly explored. This is notably the case for the two diploid species, Brassica oleracea L. (CC, 2n=2x=18) and B. rapa L. (AA, 2n=2x=20). In order to explore genetic diversity in both species, we have collected numerous populations in their center of origin, the Mediterranean basin, on a large contrasting climatic and soil gradient from northern Europe to southern sub-Saharan regions. In these areas, we also collected 14 populations belonging to five B. oleracea closely related species. Before further genetic and agronomic investigations, we controlled the absence of species misidentification using flow cytometry, sequencing of species specific chloroplast genomic region, as well as cytogenetic analyses in case of unexpected results. Looking at the 102 B. oleracea and 146 B. rapa populations showing a good germination among the 112 and 154 populations collected, seventeen populations were misidentified. The most frequent mistake was a confusion of these diploid species with B. napus . Additionally for B. rapa , 2 autotetraploid populations were observed. Habitats of the collected wild populations and landraces are described in our work. This provides a unique plant material characterization that will pave the way for further analyses investigating the genomic regions involved in climatic and microbiota adaptation. This research is supported by the H2020 Prima project ‘BrasExplor’.

Knowledge concerning the integration of genetic pathways mediating the responses to environmental cues controlling flowering initiation in crops is scarce. Here, we reveal the diversity in oilseed rape (OSR) flowering response to high ambient temperature. Using a set of different spring OSR varieties, we found a consistent flowering delay at elevated temperatures. Remarkably, one of the varieties assayed exhibited the opposite behaviour. Several FT-like paralogs are plausible candidates to be part of the florigen in OSR. We revealed that BnaFTA2 plays a major role in temperature-dependent flowering initiation. Analysis of the H2A.Z histone variant occupancy at this locus in different Brassica napus varieties produced contrasting results, suggesting the involvement of additional molecular mechanisms in BnaFTA2 repression at high ambient temperature. Moreover, BnARP6 RNAi plants showed little accumulation of H2A.Z at high temperature while maintaining temperature sensitivity and delayed flowering. Furthermore, we found that H3K4me3 present in BnaFTA2 under inductive flowering conditions is reduced at high temperature, suggesting a role for this hallmark of transcriptionally active chromatin in the OSR flowering response to warming. Our work emphasises the plasticity of flowering responses in B. napus and offers venues to optimise this process in crop species grown under suboptimal environmental conditions.

Chloroplast biogenesis is crucial in plant development, as it is essential for the transition to autotrophic growth. This process is light-induced and relies on the orchestrated transcription of nuclear and plastid genes, enabling the effective assembly and regulation of the photosynthetic machinery. Here we reveal a new regulation level for this process by showing the involvement of chromatin remodelling in the nuclear control of plastid gene expression for proper chloroplast biogenesis and function. The two Arabidopsis homologues of yeast EPL1 protein, components of the NuA4 histone acetyltransferase complex, are essential for plastid transcription and correct chloroplast development and performance. We show that EPL1 proteins are light-regulated and necessary for concerted expression of nuclear genes encoding most components of chloroplast transcriptional machinery, directly mediating H4K5ac deposition at these loci and promoting the expression of plastid genes required for chloroplast biogenesis. These data unveil a NuA4-mediated mechanism regulating chloroplast biogenesis that links the transcription of nuclear and plastid genomes during chloroplast development.

Epigenetic regulation is necessary for optimal organism development and preservation of gene expression profiles in the cell. In plants, the trimethylation of histone H3 lysine 27 (H3K27me3) is a silencing epigenetic mark relevant for developmental transitions like flowering. The floral transition is a key agronomic trait; however, the epigenetic mechanisms of flowering time regulation in crops remain poorly understood. Here we study the Jumonji H3K27me3 demethylases BraA.REF6 and BraA.ELF6 in Brassica rapa. Phenotypic characterization of novel mutant lines and genome-wide H3K27me3 chromatin immunoprecipitation and transcriptomic analyses indicated that BraA.REF6 plays a greater role than BraA.ELF6 in fine-tuning H3K27me3 levels. In addition, we found that braA.elf6 mutants were early flowering due to high H3K27me3 levels at B. rapa homologs of the floral repressor FLC. Unlike mutations in Arabidopsis thaliana, braA.ref6 mutants were late flowering without altering the expression of B. rapa FLC genes. Remarkably, we found that BraA.REF6 regulated a number of gibberellic acid (GA) biosynthetic genes, including a homolog of GA1, and that GA-treatment complemented the late flowering mutant phenotype. This study increases our understanding of the epigenetic regulation of flowering time in B. rapa, highlighting conserved and distinct regulatory mechanisms between model and crop species.

Plants react to environmental challenges by integrating external cues with endogenous signals to optimize survival and reproductive success. However, the mechanisms underlying this integration remain obscure. While stress conditions are known to impact plant development, how developmental transitions influence responses to adverse conditions has not been addressed. Here, we reveal a molecular mechanism of stress response attenuation during the onset of flowering in Arabidopsis (Arabidopsis thaliana). We show that Arabidopsis MORF-RELATED GENE (MRG) proteins, components of the NuA4 histone acetyltransferase complex that bind trimethylated-lysine 36 in histone H3 (H3K36me3), function as a chromatin switch on the floral integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) to coordinate flowering initiation with plant responsiveness to hostile environments. MRG proteins are required to activate SOC1 expression during flowering induction by promoting histone H4 acetylation. In turn, SOC1 represses a broad array of genes that mediate abiotic stress responses. We propose that during the transition from vegetative to reproductive growth, the MRG-SOC1 module constitutes a central hub in a mechanism that tunes down stress responses to enhance the reproductive success and plant fitness at the expense of costly efforts for adaptation to challenging environments.

Chromatin remodeling plays a key role in the establishment and maintenance of gene expression patterns essential for plant development and responses to environmental factors. Post-translational modification of histones, including acetylation, is one of the most relevant chromatin remodeling mechanisms that operate in eukaryotic cells. Histone acetylation is an evolutionarily conserved chromatin signature commonly associated with transcriptional activation. Histone acetylation levels are tightly regulated through the antagonistic activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In plants, different families of HATs are present, including the MYST family, which comprises homologs of the catalytic subunit of the Nucleosome Acetyltransferase of H4 (NuA4) complex in yeast. This complex mediates acetylation of histones H4, H2A, and H2A.Z, and is involved in transcriptional regulation, heterochromatin silencing, cell cycle progression, and DNA repair in yeast. In Arabidopsis and, other plant species, homologs for most of the yeast NuA4 subunits are present and although the existence of this complex has not been demonstrated yet, compelling evidence supports the notion that this type of HAT complex functions from mosses to angiosperms. Recent proteomic studies show that several Arabidopsis homologs of NuA4 components, including the assembly platform proteins and the catalytic subunit, are associated in vivo with additional members of this complex suggesting that a NuA4-like HAT complex is present in plants. Furthermore, the functional characterization of some Arabidopsis NuA4 subunits has uncovered the involvement of these proteins in the regulation of different plant biological processes. Interestingly, for most of the mutant plants deficient in subunits of this complex characterized so far, conspicuous defects in flowering time are observed, suggesting a role for NuA4 in the control of this plant developmental program. Moreover, the participation of Arabidopsis NuA4 homologs in other developmental processes, such as gametophyte development, as well as in cell proliferation and stress and hormone responses, has also been reported. In this review, we summarize the current state of knowledge on plant putative NuA4 subunits and discuss the latest progress concerning the function of this chromatin modifying complex.

SummaryFlowering time is a relevant agronomic trait because is crucial for the optimal formation of seeds and fruits. The genetic pathways controlling this developmental phase transition have been studied extensively in Arabidopsis thaliana. These pathways converge in a small number of genes including FT, the so‐called florigen, which integrates environmental cues like ambient temperature. Nevertheless, detailed and functional studies about flowering time in Brassica crops are scarce. Here we study the role of the FT Brassica rapa homologues and the effect of high ambient temperature on flowering time in this crop. Phenotypic characterization and gene‐expression analyses suggest that BraA.FT.a (BraA02g016700.3C) is decisive for initiating floral transition; consequently, braA.ft.a loss‐of‐function and hypomorphic mutations result in late flowering phenotypes. We also show that high ambient temperature delays B. rapa floral transition by reducing BraA.FT.a expression. Strikingly, these expression changes are associated with increased histone H2A.Z levels and less accessible chromatin configuration of the BraA.FT.a locus at high ambient temperature. Interestingly, increased H2A.Z levels at high ambient temperature were also observed for other B. rapa temperature‐responsive genes. Previous reports delimited that Arabidopsis flowers earlier at high ambient temperature due to reduced H2A.Z incorporation in the FT locus. Our data reveal a conserved chromatin‐mediated mechanism in B. rapa and Arabidopsis in which the incorporation of H2A.Z at FT chromatin in response to warm ambient temperature results in different flowering time responses. This work will help to develop improved Brassica crop varieties with flowering time requirements to cope with global warming.Open Research BadgesThis article has earned an Open Materials Badge for making publicly available the components of the research methodology needed to reproduce the reported procedure and analysis. Methods are available at protocols.iodx.doi.org/10.17504/protocols.io.zmff43n.

Posttranslational histone modifications and the dynamics of histone variant H2A.Z are key mechanisms underlying the floral transition. In yeast, SWR1-C and NuA4-C mediate the deposition of H2A.Z and the acetylation of histone H4, H2A and H2A.Z, respectively. Yaf9 is a subunit shared by both chromatin-remodeling complexes. The significance of the two Arabidopsis YAF9 homologues, YAF9A and YAF9B, is unknown. To get an insight into the role of Arabidopsis YAF9 proteins in plant developmental responses, we followed physiological, genetic, genomic, epigenetic, proteomics and cell biology approaches. Our data revealed that YAF9A and YAF9B are histone H3 readers with unequally redundant functions. Double mutant yaf9a yaf9b plants display pleiotropic developmental phenotypic alterations as well as misregulation of a wide variety of genes. We demonstrated that YAF9 proteins regulate flowering time by both FLC-dependent and independent mechanisms that work in parallel with SWR1-C. Interestingly, we show that YAF9A binds FLC chromatin and that YAF9 proteins regulate FLC expression by modulating the acetylation levels of H2A.Z and H4 but not H2A.Z deposition. Our work highlights the key role exerted by YAF9 homologues in the posttranslational modification of canonical histones and variants that regulate gene expression in plants to control development.

The control of precursor-messenger RNA (pre-mRNA) splicing is emerging as an important layer of regulation in plant responses to endogenous and external cues. In eukaryotes, pre-mRNA splicing is governed by the activity of a large ribonucleoprotein machinery, the spliceosome, whose protein core is composed of the Sm ring and the related Sm-like 2-8 complex. Recently, the Arabidopsis (Arabidopsis thaliana) Sm-like 2-8 complex has been characterized. However, the role of plant Sm proteins in pre-mRNA splicing remains largely unknown. Here, we present the functional characterization of Sm protein E1 (SME1), an Arabidopsis homolog of the SME subunit of the eukaryotic Sm ring. Our results demonstrate that SME1 regulates the spliceosome activity and that this regulation is controlled by the environmental conditions. Indeed, depending on the conditions, SME1 ensures the efficiency of constitutive and alternative splicing of selected pre-mRNAs. Moreover, missplicing of most targeted pre-mRNAs leads to the generation of nonsense-mediated decay signatures, indicating that SME1 also guarantees adequate levels of the corresponding functional transcripts. In addition, we show that the selective function of SME1 in ensuring appropriate gene expression patterns through the regulation of specific pre-mRNA splicing is essential for adequate plant development and adaptation to freezing temperatures. These findings reveal that SME1 plays a critical role in plant development and interaction with the environment by providing spliceosome activity specificity.

Pollen development is a crucial step in higher plants, which not only makes possible plant fertilization and seed formation, but also determines fruit quality and yield in crop species. Here, we reported a tomato T-DNA mutant, pollen deficient1 (pod1), characterized by an abnormal anther development and the lack of viable pollen formation, which led to the production of parthenocarpic fruits. Genomic analyses and the characterization of silencing lines proved that pod1 mutant phenotype relies on the tomato SlMED18 gene encoding the subunit 18 of Mediator multi-protein complex involved in RNA polymerase II transcription machinery. The loss of SlMED18 function delayed tapetum degeneration, which resulted in deficient microspore development and scarce production of viable pollen. A detailed histological characterization of anther development proved that changes during microgametogenesis and a significant delay in tapetum degeneration are associated with a high proportion of degenerated cells and, hence, should be responsible for the low production of functional pollen grains. Expression of pollen marker genes indicated that SlMED18 is essential for the proper transcription of a subset of genes specifically required to pollen formation and fruit development, revealing a key role of SlMED18 in male gametogenesis of tomato. Additionally, SlMED18 is able to rescue developmental abnormalities of the Arabidopsis med18 mutant, indicating that most biological functions have been conserved in both species.