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Background Chromatin provides a tunable platform for gene expression control. Besides the well-studied core nucleosome, H1 linker histones are abundant chromatin components with intrinsic potential to influence chromatin function. Well studied in animals, little is known about the evolution of H1 function in other eukaryotic lineages for instance plants. Notably, in the model plant Arabidopsis , while H1 is known to influence heterochromatin and DNA methylation, its contribution to transcription, molecular, and cytological chromatin organization remains elusive. Results We provide a multi-scale functional study of Arabidopsis linker histones. We show that H1-deficient plants are viable yet show phenotypes in seed dormancy, flowering time, lateral root, and stomata formation—complemented by either or both of the major variants. H1 depletion also impairs pluripotent callus formation. Fine-scale chromatin analyses combined with transcriptome and nucleosome profiling reveal distinct roles of H1 on hetero- and euchromatin: H1 is necessary to form heterochromatic domains yet dispensable for silencing of most transposable elements; H1 depletion affects nucleosome density distribution and mobility in euchromatin, spatial arrangement of nanodomains, histone acetylation, and methylation. These drastic changes affect moderately the transcription but reveal a subset of H1-sensitive genes. Conclusions H1 variants have a profound impact on the molecular and spatial (nuclear) chromatin organization in Arabidopsis with distinct roles in euchromatin and heterochromatin and a dual causality on gene expression. Phenotypical analyses further suggest the novel possibility that H1-mediated chromatin organization may contribute to the epigenetic control of developmental and cellular transitions.

Genetic transformation of many plant species relies on in vitro tissue culture-based approaches. This can be a labor-intensive process, requiring aseptic conditions and regenerating often recalcitrant species from tissue culture. Here, we have optimized an in planta transformation protocol to rapidly transform commercial citrus cultivars, bypassing the need for tissue culture. As a proof of concept, we used in planta transformation to introduce CRISPR/Cas9 constructs into Limoneira 8A Lisbon lemon and Pineapple sweet orange, cultivars that are challenging to transform with conventional techniques. Using our optimized protocol, the regeneration rate was significantly increased from 4.8% to over 95%, resulting in multiple gene-edited lines in lemon. We also successfully recovered gene-edited Pineapple sweet orange lines using this protocol; the transformation efficiency for these cultivars ranged between 0.63% and 4.17%. Remarkably, these lines were obtained within three months, making this in planta protocol a rapid methodology to obtain transformed citrus plants. This approach can rapidly and effectively introduce key genetic changes into a wide variety of citrus cultivars.

The chromatin modification H3K27me3 is involved in almost every developmental stage in Arabidopsis. Much remains unknown about the dynamic regulation of this histone modification in flower development and control of self-fertility. Here we demonstrate that the H3K27me3-specific demethylases ELF6 and JMJ13 antagonistically regulate carpel and stamen growth and thus modulate self-fertility. Transcriptome and epigenome data are used to identify potential targets of ELF6 and JMJ13 responsible for these physiological functions. We find that ELF6 relieves expansin genes of epigenetic silencing to promote cell elongation in the carpel, enhancing carpel growth and therefore encouraging out-crossing. On the other hand, JMJ13 activates genes of the jasmonic acid regulatory network alongside the auxin responsive SAUR26, to inhibit carpel growth, enhance stamen growth, and overall promote self-pollination. Our evidence provides novel mechanisms of self-fertility regulation in A. thaliana demonstrating how chromatin modifying enzymes govern the equilibrium between flower self-pollination and out-crossing.

CRISPR/Cas9 - mediated gene editing requires high efficiency to be routinely implemented, especially in species which are laborious and slow to transform. This requirement intensifies further when targeting multiple genes simultaneously, which is required for genetic screening or more complex genome engineering. Species in the Citrus genus fall into this category. Here we describe a series of experiments with the collective aim of improving multiplex gene editing in the Carrizo citrange cultivar using tRNA-based sgRNA arrays. We evaluate a range of promoters for their efficacy in such experiments and achieve significant improvements by optimizing the expression of both the Cas9 endonuclease and the sgRNA array. In the case of the former we find the UBQ10 or RPS5a promoters from Arabidopsis driving the zCas9i endonuclease variant useful for achieving high levels of editing. The choice of promoter expressing the sgRNA array also had a large impact on gene editing efficiency across multiple targets. In this respect Pol III promoters perform especially well, but we also demonstrate that the UBQ10 and ES8Z promoters from Arabidopsis are robust alternatives. Ultimately, this study provides a quantitative insight into CRISPR/Cas9 vector design that has practical application in the simultaneous editing of multiple genes in Citrus , and potentially other eudicot plant species.

Cocoa self-compatibility is an important yield factor and has been described as being controlled by a late gameto-sporophytic system expressed only at the level of the embryo sac. It results in gametic non-fusion and involves several loci. In this work, we identified two loci, located on chromosomes 1 and 4 (CH1 and CH4), involved in cocoa self-incompatibility by two different processes. Both loci are responsible for gametic selection, but only one (the CH4 locus) is involved in the main fruit drop. The CH1 locus acts prior to the gamete fusion step and independently of the CH4 locus. Using fine-mapping and genome-wide association studies, we focused analyses on restricted regions and identified candidate genes. Some of them showed a differential expression between incompatible and compatible reactions. Immunolocalization experiments provided evidence of CH1 candidate genes expressed in ovule and style tissues. Highly polymorphic simple sequence repeat (SSR) diagnostic markers were designed in the CH4 region that had been identified by fine-mapping. They are characterized by a strong linkage disequilibrium with incompatibility alleles, thus allowing the development of efficient diagnostic markers predicting self-compatibility and fruit setting according to the presence of specific alleles or genotypes. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified, thus allowing screening for self-compatible plants in cocoa populations.

Cocoa self-compatibility is an important yield factor and has been described as being controlled by a late gameto-sporophytic system expressed only at the level of the embryo sac. It results in gametic non-fusion and involves several loci. In this work, we identified two loci, located on chromosomes 1 and 4 (CH1 and CH4), involved in cocoa self-incompatibility by two different processes. Both loci are responsible for gametic selection, but only one (the CH4 locus) is involved in the main fruit drop. The CH1 locus acts prior to the gamete fusion step and independently of the CH4 locus. Using fine-mapping and genome-wide association studies, we focused analyses on restricted regions and identified candidate genes. Some of them showed a differential expression between incompatible and compatible reactions. Immunolocalization experiments provided evidence of CH1 candidate genes expressed in ovule and style tissues. Highly polymorphic simple sequence repeat (SSR) diagnostic markers were designed in the CH4 region that had been identified by fine-mapping. They are characterized by a strong linkage disequilibrium with incompatibility alleles, thus allowing the development of efficient diagnostic markers predicting self-compatibility and fruit setting according to the presence of specific alleles or genotypes. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified, thus allowing screening for self-compatible plants in cocoa populations.

Chromatin in eukaryotes provides a tunable platform to control gene expression and convey an epigenetic memory throughout cell divisions. H1 linker histones are abundant components with an intrinsic potential in influencing chromatin structure and function. We detail the impact of H1 depletion in Arabidopsis on fine-scale chromatin organization, transcription and development. While required for chromocenter assembly, H1s are dispensable for transposable element (TE) silencing and peripheral positioning of heterochromatin. In euchromatin, H1 regulates nucleosome density, mobility, and regular distribution of nanoscale chromatin domains. While necessary to maintain epigenetic patterns, H1 only moderately affects transcription. Its depletion is associated with failures in transitional fate changes such as lateral root initiation, root hair production, stomata patterning but also flowering and dormancy regulation. Therefore, Arabidopsis H1 variants are chromatin architects mediating nano- and microscale levels-of-organization operating downstream of epigenetic and transcriptional establishment processes and contribute to epigenetic reorientations in developmental transitions.

In plants, genome stability is maintained during DNA replication by the H3.1K27 methyltransferases ATXR5 and ATXR6, which catalyze the deposition of K27me1 on replication-dependent H3.1 variants. Loss of H3.1K27me1 in atxr5 atxr6 double mutants leads to heterochromatin defects, including transcriptional de-repression and genomic instability, but the molecular mechanisms involved remain largely unknown. In this study, we identified the conserved histone acetyltransferase GCN5 as a mediator of transcriptional de-repression and genomic instability in the absence of H3.1K27me1. GCN5 is part of a SAGA-like complex in plants that requires ADA2b and CHR6 to mediate the heterochromatic defects of atxr5 atxr6 mutants. Our results show that Arabidopsis GCN5 acetylates multiple lysine residues on H3.1 variants in vitro, but that H3.1K27 and H3.1K36 play key roles in inducing genomic instability in the absence of H3.1K27me1. Overall, this work reveals a key molecular role for H3.1K27me1 in maintaining genome stability by restricting histone acetylation in plants. Competing Interest Statement The authors have declared no competing interest.