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Hormones, such as auxin and cytokinin, are involved in the complex molecular network that regulates the coordinated development of plant organs. Genes controlling ovule patterning have been identified and studied in detail; however, the roles of auxin and cytokinin in ovule development are largely unknown. Here we show that key cytokinin pathway genes, such as isopentenyltransferase and cytokinin receptors, are expressed during ovule development. Also, in a cre1-12 ahk2-2 ahk3-3 triple mutant with severely reduced cytokinin perception, expression of the auxin efflux facilitator PIN-FORMED 1 (PIN1) was severely reduced. In sporocyteless/nozzle (spl/nzz) mutants, which show a similar phenotype to the cre1-12 ahk2-2 ahk3-3 triple mutant, PIN1 expression is also reduced. Treatment with the exogenous cytokinin N 6 -benzylaminopurine also altered both auxin distribution and patterning of the ovule; this process required the homeodomain transcription factor BELL1 (BEL1). Thus, this article shows that cytokinin regulates ovule development through the regulation of PIN1. Furthermore, the transcription factors BEL1 and SPL/NZZ, previously described as key regulators of ovule development, are needed for the auxin and cytokinin signaling pathways for the correct patterning of the ovule.

The phytohormone auxin governs crucial developmental decisions throughout the plant life cycle. Auxin signaling is effectuated by auxin response factors (ARFs) whose activity is repressed by Aux/IAA proteins under low auxin levels, but relieved from repression when cellular auxin concentrations increase. ARF3/ETTIN (ETT) is a conserved noncanonical Arabidopsis thaliana ARF that adopts an alternative auxin-sensing mode of translating auxin levels into multiple transcriptional outcomes. However, a mechanistic model for how this auxin-dependent modulation of ETT activity regulates gene expression has not yet been elucidated. Here, we take a genome-wide approach to show how ETT controls developmental processes in the Arabidopsis shoot through its auxin-sensing property. Moreover, analysis of direct ETT targets suggests that ETT functions as a central node in coordinating auxin dynamics and plant development and reveals tight feedback regulation at both the transcriptional and protein-interaction levels. Finally, we present an example to demonstrate how auxin sensitivity of ETT-protein interactions can shape the composition of downstream transcriptomes to ensure specific developmental outcomes. These results show that direct effects of auxin on protein factors, such as ETT-TF complexes, comprise an important part of auxin biology and likely contribute to the vast number of biological processes affected by this simple molecule.

Specification of new organs from transit amplifying cells is critical for higher eukaryote development. In plants, a central stem cell pool maintained by the pluripotency factor SHOOTMERISTEMLESS (STM), is surrounded by transit amplifying cells competent to respond to auxin hormone maxima by giving rise to new organs. Auxin triggers flower initiation through Auxin Response Factor (ARF) MONOPTEROS (MP) and recruitment of chromatin remodelers to activate genes promoting floral fate. The contribution of gene repression to reproductive primordium initiation is poorly understood. Here we show that downregulation of the STM pluripotency gene promotes initiation of flowers and uncover the mechanism for STM silencing. The ARFs ETTIN (ETT) and ARF4 promote organogenesis at the reproductive shoot apex in parallel with MP via histone-deacetylation mediated transcriptional silencing of STM . ETT and ARF4 directly repress STM , while MP acts indirectly, through its target FILAMENTOUS FLOWER ( FIL ). Our data suggest that – as in animals- downregulation of the pluripotency program is important for organogenesis in plants. The pluripotency factor SHOOTMERISTEMLESS (STM) maintains stem cell identity in the centre of the shoot apical meristem. Here Chung et al. show that Auxin Response Factors work in concert with the YABBY transcription factor FIL to silence STM via chromatin modifications and allow initiation of primordia that lead to formation of flowers.

The BASIC PENTACYSTEINE (BCP) family is a poorly characterized plant transcription factor family of GAGA BINDING PROTEINS. In Arabidopsis, there are seven members (BPC1–7) that are broadly expressed, and they can potentially bind more than 3000 Arabidopsis GAGA-repeat-containing genes. To date, BPCs are known to be direct regulators of the INNER NO OUTER (INO), SEEDSTICK (STK), and LEAFY COTYLEDON 2 (LEC2) genes. Because of the high functional redundancy, neither single knockout nor double bpc mutant combinations cause aberrant phenotypes. The bpc1-2 bpc2 bpc3 triple mutant shows several pleiotropic developmental defects, including enlargement of the inflorescence meristem and flowers with supernumerary floral organs. Here, we demonstrated through expression analysis and chromatin immunoprecipitation assays that this phenotype is probably due to deregulation of the expression of the SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS/KNAT1 (BP) genes, which are both direct targets of BPCs. Moreover, we assigned a role to BPCs in the fine regulation of the cytokinin content in the meristem, as both ISOPENTENYLTRANSFERASE 7 (IPT7) and ARABIDOPSIS RESPONSE REGULATOR 7 (ARR7) genes were shown to be overexpressed in the bpc1-2 bpc2 bpc3 triple mutant.

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. We profiled the genome-wide distribution of three histone modifications, H3K27me3, H3K4me3, and H3K27Ac, from a few seedlings of 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. CUT&Tag is a valid, easy-to-perform, cost-effective, and reliable approach for efficient epigenomic profiling in , 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.

BASIC PENTACYSTEINE (BPC) transcription factors have been identified in a large variety of plant species. In Arabidopsis thaliana there are seven BPC genes, which, except for BPC5, are expressed ubiquitously. BPC genes are functionally redundant in a wide range of developmental processes. Recently, we reported that BPC1 binds to guanine and adenine (GA)-rich consensus sequences in the SEEDSTICK (STK) promoter in vitro and induces conformational changes. Here we show by chromatin immunoprecipitation experiments that in vivo BPCs also bind to the consensus boxes, and when these were mutated, expression from the STK promoter was derepressed, resulting in ectopic expression in the inflorescence. We also reveal that SHORT VEGETATIVE PHASE (SVP) is a direct regulator of STK. SVP is a floral meristem identity gene belonging to the MADS box gene family. The SVP-APETALA1 (AP1) dimer recruits the SEUSS (SEU)-LEUNIG (LUG) transcriptional cosuppressor to repress floral homeotic gene expression in the floral meristem. Interestingly, we found that GA consensus sequences in the STK promoter to which BPCs bind are essential for recruitment of the corepressor complex to this promoter. Our data suggest that we have identified a new regulatory mechanism controlling plant gene expression that is probably generally used, when considering BPCs' wide expression profile and the frequent presence of consensus binding sites in plant promoters.

The plant hormone auxin regulates numerous aspects of the plant life cycle. Auxin signalling is mediated by auxin response factors (ARFs) that dimerise with modulating Aux/IAA repressors. ARF3 (ETTIN or ETT) is atypical as it does not interact with Aux/IAA repressors. It is proposed to be a non-canonical auxin sensor, regulating diverse functions essential for development. This sensing ability relies on a unique C-terminal ETT specific domain (ES domain). Alignments of ETT orthologues across the angiosperm phylum revealed that the length and sequence identities of ES domains are poorly conserved. Computational predictors suggested the ES domains to be intrinsically disordered, explaining their tolerance of insertions, deletions and mutations during evolution. Nevertheless, five highly conserved short linear motifs were identified suggesting functional significance. High-throughput library screening identified an almost full-length soluble ES domain that did not bind auxin directly, but exhibited a dose-dependent response in a yeast two-hybrid system against the Arabidopsis INDEHISCENT (IND) transcription factor. Circular dichroism confirmed the domain was disordered. The identification and purification of this domain opens the way to the future characterisation of the ETT auxin-sensing mechanism in planta and an improved understanding of auxin-mediated regulation.

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. 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. 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 specialized, differentiated cells, the gametes. At maturity, male and female gametes are quiescent, awaiting fertilization, with their cell cycle being arrested at a precise stage. Failure to establish quiescence leads to unwanted proliferation, abortion of the offspring, and a waste of resources. Upon fertilization, the cell cycle resumes, allowing the newly formed zygote to divide rapidly. Successful development requires that male and female gametes are in the same phase of the cell cycle. The molecular mechanisms that enforce quiescence and reinstate cell division only after fertilization occurs are poorly understood. Here, we describe a sperm-derived signal that induces proliferation of the Arabidopsis central cell precisely upon fertilization. We show that the mature central cell is arrested in S phase, caused by the activity of the conserved RETINOBLASTOMA RELATED1 (RBR1) protein. Paternal delivery of the core cell cycle component CYCD7;1 triggers RBR1 degradation, thereby stimulating S phase progression. Absence of CYCD7;1 delays RBR1 depletion, S phase reactivation, and central cell division, whereas its constitutive expression triggers proliferation of unfertilized central cells. In summary, we show that CYCD7;1 is a paternal signal that informs the central cell that fertilization occurred, thus unlocking quiescence and ensuring that cell division initiates just at the right time to ensure functional endosperm formation.Competing Interest StatementThe authors have declared no competing interest.

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.