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The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. We found that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed histone H3 lysine 27 monomethylation in plants depends on H3.1, TSK, and DNA polymerase theta (Pol θ). This work reveals an H3.1-specific function during replication and a common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone monomethyltransferases and reading of the H3.1 variant.

Transformation viaAgrobacterium tumefaciens(Agrobacterium) is the predominant method used to introduce exogenous DNA into plants. Transfer DNA (T-DNA) originating from Agrobacterium can be integrated as a single copy or in concatenated forms in plant genomes, but the mechanisms affecting final T-DNA structure remain unknown. In this study, we demonstrate that the inclusion of retrotransposon (RT)-derived sequences in T-DNA can increase transgene copy number by more than 50-fold inArabidopsis thaliana(Arabidopsis). RT-mediated amplification of T-DNA results in large concatemers in the Arabidopsis genome, which are primarily induced by the long terminal repeats (LTRs) of RTs. T-DNA amplification is dependent on the activity of DNA repair proteins associated with theta-mediated end joining (TMEJ). Finally, we show that T-DNA amplification can increase the frequency of targeted mutagenesis and gene targeting. Overall, this work uncovers molecular determinants that modulate T-DNA copy number in Arabidopsis and demonstrates the utility of inducing T-DNA amplification for plant gene editing.

Flowering of the reference legume Medicago truncatula is promoted by winter cold (vernalization) followed by long-day photoperiods (VLD) similar to winter annual Arabidopsis. However, Medicago lacks FLC and CO, key regulators of Arabidopsis VLD flowering. Most plants have two INHIBITOR OF GROWTH (ING) genes (ING1 and ING2), encoding proteins with an ING domain with two anti-parallel alpha-helices and a plant homeodomain (PHD) finger, but their genetic role has not been previously described. In Medicago, Mting1 gene-edited mutants developed and flowered normally, but an Mting2-1 Tnt1 insertion mutant and gene-edited Mting2 mutants had developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes, and smaller seeds and barrels. Mting2 mutants had reduced expression of activators of flowering, including the FT-like gene MtFTa1, and increased expression of the candidate repressor MtTFL1c, consistent with the delayed flowering of the mutant. MtING2 overexpression complemented Mting2-1, but did not accelerate flowering in wild type. The MtING2 PHD finger bound H3K4me2/3 peptides weakly in vitro, but analysis of gene-edited mutants indicated that it was dispensable to MtING2 function in wild-type plants. RNA sequencing experiments indicated that7000 genes are mis-expressed in the Mting2-1 mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP-seq analysis identified5000 novel H3K4me3 locations in the genome of Mting2-1 mutants compared to wild type R108. Overall, our mutant study has uncovered an important physiological role of a plant ING2 gene in development, flowering, and gene expression, which likely involves an epigenetic mechanism.

Background The three-dimensional nuclear arrangement of chromatin impacts many cellular processes operating at the DNA level in animal and plant systems. Chromatin organization is a dynamic process that can be affected by biotic and abiotic stresses. Three-dimensional imaging technology allows to follow these dynamic changes, but only a few semi-automated processing methods currently exist for quantitative analysis of the 3D chromatin organization. Results We present an automated method, Nuclear Object DetectionJ (NODeJ), developed as an imageJ plugin. This program segments and analyzes high intensity domains in nuclei from 3D images. NODeJ performs a Laplacian convolution on the mask of a nucleus to enhance the contrast of intra-nuclear objects and allow their detection. We reanalyzed public datasets and determined that NODeJ is able to accurately identify heterochromatin domains from a diverse set of Arabidopsis thaliana nuclei stained with DAPI or Hoechst. NODeJ is also able to detect signals in nuclei from DNA FISH experiments, allowing for the analysis of specific targets of interest. Conclusion and availability NODeJ allows for efficient automated analysis of subnuclear structures by avoiding the semi-automated steps, resulting in reduced processing time and analytical bias. NODeJ is written in Java and provided as an ImageJ plugin with a command line option to perform more high-throughput analyses. NODeJ can be downloaded from https://gitlab.com/axpoulet/image2danalysis/-/releases with source code, documentation and further information avaliable at https://gitlab.com/axpoulet/image2danalysis. The images used in this study are publicly available at https://www.brookes.ac.uk/indepth/images/ and https://doi.org/10.15454/1HSOIE.

Optimizing flowering time is crucial for maximizing crop productivity, but gaps remain in the knowledge of the mechanisms underpinning temperate legume flowering. Medicago, like winter annual Arabidopsis, accelerates flowering after exposure to extended cold (vernalization, V) followed by long-day (LD) photoperiods. In Arabidopsis, photoperiodic flowering is triggered through CO, a photoperiodic switch that directly activates the FT gene encoding a mobile florigen and potent activator of flowering. In Arabidopsis, several CYCLING DOF FACTORs (CDFs), including AtCDF1, act redundantly to repress CO and thus FT expression, until their removal in LD by a blue-light-induced F-BOX1/GIGANTEA (FKF1/GI) complex. Medicago possesses a homolog of FT, MtFTa1, which acts as a strong activator of flowering. However, the regulation of MtFTa1 does not appear to involve a CO-like gene. Nevertheless, work in pea suggests that CDFs may still regulate flowering time in temperate legumes. Here, we analyze the function of Medicago MtCDF genes with a focus on MtCDFd1_1 in flowering time and development. MtCDFd1_1 causes strong delays to flowering when overexpressed in Arabidopsis and shows a cyclical diurnal expression in Medicago with peak expression at dawn, consistent with AtCDF genes like AtCDF1. However, MtCDFd1_1 lacks predicted GI or FKF1 binding domains, indicating possible differences in its regulation from AtCDF1. In Arabidopsis, CDFs act in a redundant manner, and the same is likely true of temperate legumes as no flowering time phenotypes were observed when MtCDFd1_1 or other MtCDFs were knocked out in Medicago Tnt1 lines. Nevertheless, overexpression of MtCDFd1_1 in Medicago plants resulted in late flowering relative to wild type in inductive vernalized long-day (VLD) conditions, but not in vernalized short days (VSDs), rendering them day neutral. Expression of MtCO-like genes was not affected in the transgenic lines, but LD-induced genes MtFTa1, MtFTb1, MtFTb2, and MtSOC1a showed reduced expression. Plants carrying both the Mtfta1 mutation and 35S:MtCDFd1_1 flowered no later than the Mtfta1 plants. This indicates that 35S:MtCDFd1_1 likely influences flowering in VLD via repressive effects on MtFTa1 expression. Overall, our study implicates MtCDF genes in photoperiodic regulation in Medicago by working redundantly to repress FT-like genes, particularly MtFTa1, but in a CO-independent manner, indicating differences from the Arabidopsis model.

Optimising the timing of flowering contributes to successful sexual reproduction and yield in agricultural plants. FLOWERING LOCUS T (FT) genes, first identified in Arabidopsis thaliana (Arabidopsis), promote flowering universally, but the upstream flowering regulatory pathways can differ markedly among plants. Flowering in the model legume, Medicago truncatula (Medicago) is accelerated by winter cold (vernalisation) followed by long day (LD) photoperiods leading to elevated expression of the floral activator, FT-like gene FTa1. However, Medicago, like some other plants, lacks the activator CONSTANS (CO) and the repressor FLOWERING LOCUS C (FLC) genes which directly regulate FT and are key to LD and vernalisation responses in Arabidopsis. Conversely, Medicago has a VERNALISATION2-LIKE VEFS-box gene (MtVRN2). In Arabidopsis AtVRN2 is a key member of a Polycomb complex involved in stable repression of Arabidopsis FLC after vernalisation. VRN2-like genes have been identified in other eudicot plants, but their function has never been reported. We show that Mtvrn2 mutants bypass the need for vernalisation for early flowering in LD conditions in Medicago. Investigation of the underlying mechanism by transcriptome analysis reveals that Mtvrn2 mutants precociously express FTa1 and other suites of genes including floral homeotic genes. Double-mutant analysis indicates that early flowering is dependent on functional FTa1. The broad significance of our study is that we have demonstrated a function for a VRN2-like VEFS gene beyond the Brassicaceae. In particular, MtVRN2 represses the transition to flowering in Medicago by regulating the onset of expression of the potent floral activator, FTa1.

Photoperiodic flowering aligns plant reproduction to favourable seasons of the year to maximise successful production of seeds and grains. However understanding of this process in the temperate legumes of the Fabaceae family, which are important both agriculturally and ecologically, is incomplete. Previous work in the reference legume Medicago truncatula has shown that the FT-like gene MtFTa1 is a potent floral activator. While MtFTa1 is upregulated by long-day photoperiods (LD) and vernalisation, the molecular basis of this is unknown as functional homologues of key regulatory genes present in other species, notably CONSTANS in A. thaliana, have not been identified. In LD MtFTa1 maintains a near constant diurnal pattern of expression unlike its homologue FT in A. thaliana, which has a notable peak in expression at dusk. This suggests a different manner of regulation. Furthermore, M. truncatula possesses other FT-like genes such as two LD induced MtFTb genes which may also act in the regulation of flowering time. MtFTb genes have a diurnal pattern of expression with peaks at both four and sixteen hours after dawn. This study utilises RNA-Seq to analyse the transcriptome of M. truncatula leaves to identify genes which may regulate or be co-expressed with these FT-like genes following a shift from short-day photoperiods to inductive long-days. Specifically this study focuses on the first four hours of the day in the young leaves, which coincides with the first diurnal peak of the FTb genes. Following differential expression analysis at each timepoint, genes which alter their pattern of expression are distinguished from those which just alter their magnitude of expression (and those that do neither). It goes on to categorise these genes into groups with similar patterns of expression using c-means clustering and identifies a number of potential candidate photoperiod flowering time genes for future studies to consider.

Background The glycolytic pathway is central to cellular energy production. Selection on individual enzymes within glycolysis, particularly phosphoglucose isomerase (Pgi), has been associated with metabolic performance in numerous organisms. Nonetheless, how whole energy-producing pathways evolve to allow organisms to thrive in different environments and adopt new lifestyles remains little explored. The Lanceocercata radiation of Australasian stick insects includes transitions from tropical to temperate climates, lowland to alpine habitats, and winged to wingless forms. This permits a broad investigation to determine which steps within glycolysis and what sites within enzymes are the targets of positive selection. To address these questions we obtained transcript sequences from seven core glycolysis enzymes, including two Pgi paralogues, from 29 Lanceocercata species. Results Using maximum likelihood methods a signature of positive selection was inferred in two core glycolysis enzymes. Pgi and Glyceraldehyde 3-phosphate dehydrogenase (Gaphd) genes both encode enzymes linking glycolysis to the pentose phosphate pathway. Positive selection among Pgi paralogues and orthologues predominately targets amino acids with residues exposed to the protein’s surface, where changes in physical properties may alter enzyme performance. Conclusion Our results suggest that, for Lancerocercata stick insects, adaptation to new stressful lifestyles requires a balance between maintaining cellular energy production, efficiently exploiting different energy storage pools and compensating for stress-induced oxidative damage.