Sara Simonini

Development of multicellular organisms requires the precise coordination of gene expression programs. Whereas histone modifications are key regulators of transcriptional states, how these modifications are interpreted during early development remains poorly understood. Here, we investigate the role of H3K4me3 recognition during embryogenesis in Arabidopsis thaliana . We identify ALFIN-LIKE (AL) proteins as essential H3K4me3 readers required for early seed development. Loss of AL function leads to severe defects in embryogenesis, including impaired asymmetric zygotic division, disrupted patterning, and loss of lineage restriction. At the molecular level, AL deficiency results in widespread transcriptional misregulation and is associated with local changes in chromatin state, including reduced H3K4me3 and a concomitant increase in H3K27me3 at a subset of loci, including key developmental and flowering-time regulators. Together, our results show that interpretation of H3K4me3 by AL proteins is required for proper gene expression during early development and reveal a context-dependent relationship between activating and repressive chromatin states, highlighting a role for H3K4me3 in shaping gene expression during plant embryogenesis.

Most organisms, including plants, can encode stress memories that improve resilience to repeated environmental challenges. Flooding events expose plants to recurrent hypoxia, yet whether plants establish an adaptive memory of flooding is unclear. Here we show that somatic flooding stress memory is a conserved feature across multiple angiosperm species. In Arabidopsis , this memory depends on the oxygen-sensitive Polycomb Repressive Complex 2 (PRC2) subunit VERNALIZATION2 (VRN2). Loss of VRN2 impairs epigenetic memory formation and disrupts transcriptional memory at key genes that promote anthocyanin accumulation and repress leaf senescence, adaptive responses that enhance flooding tolerance. Collectively, we reveal a molecular mechanism where VRN2-PRC2 acts as both an oxygen sensor and chromatin effector to establish adaptive flooding stress memory in plants. Short: Our work defines a molecular mechanism for flooding stress memory in plants, actioned via the PRC2 subunit VRN2, that acts as the direct molecular (low) oxygen sensor and transducer. Coupled to its role in cold sensing, this identifies VRN2 as a master sensor subunit of the plant PRC2 that integrates multiple environmental cues to modulate the chromatin landscape and encode environmental memory.

Background: The genome‐wide profiling of chromatin states that are defined by different histone posttranslational modifications, known as epigenomic profiling, is crucial for understanding the epigenetic regulations of gene expression, both in animal and plant systems. CUT&Tag (Cleavage Under Targets and Tagmentation) is a novel enzyme‐tethering method for epigenomic profiling, initially developed for mammalian cells. CUT&Tag has several advantages compared to the most commonly used epigenomic profiling methods such as chromatin immunoprecipitation followed by high‐throughput sequencing (ChIP‐seq). CUT&Tag allows epigenenomic profiling from a much smaller amount of starting material compared to ChIP‐seq. Moreover, CUT&Tag relies on in situ DNA cleavage mediated by a transposase tethered to antibodies, while ChIP‐seq typically involves nearly random DNA fragmentation. This fundamental difference raises the important question of whether CUT&Tag provides distinct advantages in terms of resolution and the precision of epigenomic profiles compared to ChIP‐seq. Results: We profiled the genome‐wide distribution of three histone modifications, H3K27me3, H3K4me3, and H3K27Ac, from a few seedlings of Arabidopsis that weighed around 0.01 g. We compared the CUT&Tag H3K27me3 profile with publicly available H3K27me3 profiles generated from ChIP‐seq, and ChIPmentation, a ChIP‐seq–based technique where MNase and Tn5 transposon are used to cleave the chromatin and prepare sequencing libraries, respectively. We showed that all these techniques capture the same broad lines of the epigenomes, but they also showed preferences toward different genomic features and revealed different sets of peaks. Analysis using the CUT&Tag datasets for the three histone modifications revealed their genomic locations and their relationship with the gene expression level, which are consistent with the expected effect of these histone marks on gene transcription. By comparing to the nucleosome occupancy data, we show that CUT&Tag reached resolution at the single‐nucleosome level, which is similar to that of ChIPmentation, but higher than that of ChIP‐seq. Moreover, both CUT&Tag and ChIPmentation data revealed an enrichment of H3K27me3 mark on exons, thus providing a deeper understanding of epigenome features that could not be resolved by ChIP‐seq. Conclusion: CUT&Tag is a valid, easy‐to‐perform, cost‐effective, and reliable approach for efficient epigenomic profiling in Arabidopsis , compatible with limited amounts of plant tissue, and provides a higher resolution compared to that of ChIP‐seq. Because the CUT&Tag protocol starting input is isolated nuclei, it is applicable to model and nonmodel plants.

SUMMARY: Floral organ identity is controlled largely by the combinatorial action of MADS domain homeotic transcription factors. Lodicules are specialized plant organs in cereals and grasses that are involved in floret opening and facilitate pollination and fertility in rice ( Oryza sativa L.). To understand the mechanisms underlying the specification of the rice lodicule, we investigated the developmental functions of the rice PISTILLATA ( PI ) paralogs, OsMADS2 , and OsMADS4 . Null osmads2 mutants reiterated OsMADS2 nonredundant lodicule specification roles and revealed new roles in flowering time and floral organ number and fate. Doubly perturbed osmads2 osmads4kd florets had severe abnormalities, were female infertile, yet could initiate parthenocarpy. Ubiquitous OsMADS4 overexpression rescued osmads2 abnormalities. We also utilized genome‐wide binding analyses and transcriptome profiling to identify putative target genes contributing to OsMADS2 functions. In osmads2 ^d8/d8 null mutant, we observed deregulated genes in a plethora of processes including lodicule and stamen development, floral organ number, and cell wall development. Some examples are cell division regulators (Cyclin D6, Cyclin‐P4‐1‐like), an aquaporin (PIP1A), a peptide transporter, a vascular developmental regulator (HOX1), and a cell wall modulator (GH9B16). The deregulation of these genes may be associated with the disrupted cell division, tissue differentiation, and physiology of the malformed lodicules in osmads2 and osmads2 osmads4kd florets. Altogether, we reveal novel roles for the rice PI paralogs in flowering time, panicle exsertion, and embryo sac differentiation, identify gene targets for lodicule development, and provide mechanistic insights on the functional diversification of rice PI paralogs.

In multicellular organisms such as animals and plants, development requires the precise regulation of gene expression, mediated not only by transcription factors but also by chromatin-based mechanisms. Among these, histone modifications like H3K4me3 and H3K27me3 play opposing roles in gene activation and repression, respectively. In Arabidopsis thaliana , H3K27me3 is deposited by the Polycomb Repressive Complex 2 (PRC2), while Trithorax group (TrxG) proteins mediate H3K4me3 deposition. While the functions of these writer complexes have been extensively studied, far less is known about the histone mark readers that interpret these modifications during development. Here, we investigate the antagonistic interplay between H3K27me3 and H3K4me3 during Arabidopsis embryogenesis. We identify a developmentally specific interaction between the FIS-PRC2 complex and ALFIN-LIKE (AL) proteins—a family of plant-specific PHD domain proteins that read H3K4me3. Our findings reveal a dynamic competition between these two marks during early embryogenesis that helps shape the epigenomic landscape of the developing seed. Disruption of AL function leads to severe developmental defects and loss of cell identity in early embryos. Moreover, loss of ALs impairs H3K4me3 deposition, resulting in aberrant spreading of H3K27me3, misregulation of developmental genes, and defects that persist into adult plant traits. Together, our results show that proper embryonic development relies on a finely tuned antagonism between activating and repressive chromatin states—an interplay orchestrated not only by their writers but also by specific readers that translate these epigenetic cues into developmental outcomes.

Cell division is a fundamental process shared across diverse life forms, from yeast to humans and plants. Multicellular organisms reproduce through the formation of specialized types of cells, the gametes, which at maturity enter a quiescent state that can last decades. At the point of fertilization, signalling lifts the quiescent state and triggers cell cycle reactivation. Studying how the cell cycle is regulated during plant gamete development and fertilization is challenging, and decades of research have provided valuable, yet sometimes contradictory, insights. This Review summarizes the current understanding of plant cell cycle regulation, gamete development, quiescence, and fertilization-triggered reactivation.

Various proteins interact with specific genome regions, playing crucial roles in gene regulation. Chromatin Immunoprecipitation (ChIP) is the most commonly used method to study protein-DNA interactions in vivo. By combining ChIP with high-throughput sequencing, ChIP-seq allows for studying the genome-wide localization of proteins. Although several ChIP protocols are available for plant tissues, they are primarily designed for histone modifications and abundant proteins with high DNA-binding affinity, which are considered as the “standard targets.” Here we describe a ChIP protocol for plant tissues not only optimized for the standard targets but also adapted for proteins with low abundances or weak DNA-binding ability. Successful execution of the protocol enables reliable generation of DNA templates for quantitative PCR or libraries for next-generation sequencing, which makes it an effective tool for analyzing genomic interactions of a wide range of proteins.

Background: The genome-wide profiling of chromatin states that are defined by different histone post-translational modifications, known as epigenomic profiling, is crucial for understanding the epigenetic regulations of gene expression, both in animal and plant systems. CUT&Tag (Cleavage Under Targets and Tagmentation, [1]) is a novel enzyme-tethering method for epigenomic profiling, initially developed for mammalian cells. CUT&Tag has several advantages compared to the most commonly used epigenomic profiling methods such as Chromatin Immunoprecipitation followed by high-throughput sequencing (ChIP-seq). CUT&Tag allows epigenenomic profiling from a much less amount of starting material compared to ChIP-seq. CUT&Tag is based on the in situ cleavage of DNA by enzymes tethered to antibodies, while in ChIP-seq, the cleavage is done by a nearly random fragmentation step. In theory, this difference in the way of cleaving DNA allows CUT&Tag to reach a higher resolution compared to ChIP-seq. Therefore, CUT&Tag holds the potential to profile the genome-wide distribution at a high resolution even from a small amount of plant tissues. Results: We profiled the genome-wide distribution of three histone modifications, H3K27me3, H3K4me3 and H3K27Ac, from a few seedlings of Arabidopsis that weighed around 0.01 grams. By comparing the H3K27me3 profiles generated from ChIP-seq and CUT&Tag, we showed that CUT&Tag and ChIP-seq capture the same broad lines of the epigenomes, but they also revealed different sets of peaks. Analysis using the CUT&Tag datasets for the three histone modifications revealed their genomic locations and their relationship with the gene expression level, which are consistent with the expected effect of these histone marks on gene transcription. By comparing to the nucleosome occupancy data, we show that CUT&Tag reached nucleosomal resolution, a much higher resolution than ChIP-seq. In the end, we presented that the increased resolution of CUT&Tag could better reveal the exon enrichment of histone modifications and the epigenetic states of the +1 nucleosome, showing benefits and advantages that this technique could bring to the field of plant epigenetics and chromatin study in general. Conclusion: CUT&Tag is a valid, easy-to-perform, cost-effective, and reliable approach for efficient epigenomic profiling in Arabidopsis , even with limited amount of starting material and provides a higher resolution compared to ChIP-seq. Because the CUT&Tag protocol starting input is isolated nuclei, it is also applicable to other model and non-model plants.

In multicellular organisms, sexual reproduction relies on the formation of highly differentiated cells, the gametes, which await fertilization in a quiescent state. Upon fertilization, the cell cycle resumes. Successful development requires that male and female gametes are in the same phase of the cell cycle. The molecular mechanisms that reinstate cell division in a fertilization-dependent manner are poorly understood in both animals and plants. Using Arabidopsis , we show that a sperm-derived signal induces the proliferation of a female gamete, the central cell, precisely upon fertilization. The central cell is arrested in S phase by the activity of the RETINOBLASTOMA RELATED1 (RBR1) protein. Upon fertilization, delivery of the core cell cycle component CYCD7;1 causes RBR1 degradation and thus S phase progression, ensuring the formation of functional endosperm and, consequently, viable seeds.

The gynoecium is critical for the reproduction of flowering plants as it contains the ovules and the tissues that foster pollen germination, growth, and guidance. These tissues, known as the reproductive tract (ReT), comprise the stigma, style, and transmitting tract (TT). The ReT and ovules originate from the carpel margin meristem (CMM) within the pistil. SHOOT MERISTEMLESS (STM) is a key transcription factor for meristem formation and maintenance. In all above-ground meristems, including the CMM, local STM downregulation is required for organ formation. However, how this downregulation is achieved in the CMM is unknown. Here, we have studied the role of HISTONE DEACETYLASE 19 (HDA19) in Arabidopsis (Arabidopsis thaliana) during ovule and ReT differentiation based on the observation that the hda19-3 mutant displays a reduced ovule number and fails to differentiate the TT properly. Fluorescence-activated cell sorting coupled with RNA-sequencing revealed that in the CMM of hda19-3 mutants, genes promoting organ development are downregulated while meristematic markers, including STM, are upregulated. HDA19 was essential to downregulate STM in the CMM, thereby allowing ovule formation and TT differentiation. STM is ectopically expressed in hda19-3 at intermediate stages of pistil development, and its downregulation by RNA interference alleviated the hda19-3 phenotype. Chromatin immunoprecipitation assays indicated that STM is a direct target of HDA19 during pistil development and that the transcription factor SEEDSTICK is also required to regulate STM via histone acetylation. Thus, we identified factors required for the downregulation of STM in the CMM, which is necessary for organogenesis and tissue differentiation.