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We describe complete design of a synthetic eukaryotic genome, Sc2.0, a highly modified Saccharomyces cerevisiae genome reduced in size by nearly 8%, with 1.1 megabases of the synthetic genome deleted, inserted, or altered. Sc2.0 chromosome design was implemented with BioStudio, an open-source framework developed for eukaryotic genome design, which coordinates design modifications from nucleotide to genome scales and enforces version control to systematically track edits. To achieve complete Sc2.0 genome synthesis, individual synthetic chromosomes built by Sc2.0 Consortium teams around the world will be consolidated into a single strain by “endoreduplication intercross.” Chemically synthesized genomes like Sc2.0 are fully customizable and allow experimentalists to ask otherwise intractable questions about chromosome structure, function, and evolution with a bottom-up design strategy.

A contribution of DNA methylation to defense against invading nucleic acids and maintenance of genome integrity is uncontested; however, our understanding of the extent of involvement of this epigenetic mark in genome-wide gene regulation and plant developmental control is incomplete. Here, we knock out all five known DNA methyltransferases in Arabidopsis , generating DNA methylation-free plants. This quintuple mutant exhibits a suite of developmental defects, unequivocally demonstrating that DNA methylation is essential for multiple aspects of plant development. We show that CG methylation and non-CG methylation are required for a plethora of biological processes, including pavement cell shape, endoreduplication, cell death, flowering, trichome morphology, vasculature and meristem development, and root cell fate determination. Moreover, we find that DNA methylation has a strong dose-dependent effect on gene expression and repression of transposable elements. Taken together, our results demonstrate that DNA methylation is dispensable for Arabidopsis survival but essential for the proper regulation of multiple biological processes. Our understanding of the extent of involvement of DNA methylation in genome-wide gene regulation and plant developmental control is incomplete. Here, the authors knock out all five known DNA methyltransferases and show the developmental and gene expression changes in the DNA methylation-free Arabidopsis plants.

In many developmental contexts, cell lineages have variable or flexible potency to self-renew. What drives a cell to exit from a proliferative state and begin differentiation, or to retain the capacity to divide days or years later is not clear. Here we exploit the mixed potential of the stomatal lineage ground cell (SLGC) in the Arabidopsis leaf epidermis as a model to explore how cells might balance potential to differentiate with a reentry into proliferation. By generating transcriptomes of fluorescence-activated cell sorting-isolated populations that combinatorically define SLGCs and integrating these data with other stomatal lineage datasets, we find that SLGCs appear poised between proliferation and endoreduplication. Furthermore, we found the RNA polymerase II-related mediator complex interactor DEK and the transcription factor MYB16 accumulate differentially in the stomatal lineage and influence the extent of cell proliferation during leaf development. These findings suggest that SLGC latent potential is maintained by poising of the cell cycle machinery, as well as general and site-specific gene-expression regulators.

The scarcity of accessible sites that are dynamic or cell type-specific in plants may be due in part to tissue heterogeneity in bulk studies. To assess the effects of tissue heterogeneity, we apply single-cell ATAC-seq to Arabidopsis thaliana roots and identify thousands of differentially accessible sites, sufficient to resolve all major cell types of the root. We find that the entirety of a cell’s regulatory landscape and its transcriptome independently capture cell type identity. We leverage this shared information on cell identity to integrate accessibility and transcriptome data to characterize developmental progression, endoreduplication and cell division. We further use the combined data to characterize cell type-specific motif enrichments of transcription factor families and link the expression of family members to changing accessibility at specific loci, resolving direct and indirect effects that shape expression. Our approach provides an analytical framework to infer the gene regulatory networks that execute plant development. Existing studies of the chromatin accessibility, the primary mark of regulatory DNA, in Arabidopsis are based mainly on bulk samples. Here, the authors report the regulatory landscape of Arabidopsis thaliana roots at single-cell resolution.

As sessile organisms, plants face a variety of environmental challenges. Their reproduction and survival depend on their ability to adapt to these stressors, which include water, heat stress, high salinity, and pathogen infection. Failure to adapt to these stressors results in programmed cell death and decreased viability, as well as reduced productivity in the case of crop plants. The growth and development of plants are maintained by meiosis and mitosis as well as endoreduplication, during which DNA replicates without cytokinesis, leading to polyploidy. As in other eukaryotes, the cell cycle in plants consists of four stages (G1, S, G2, and M) with two major check points, namely, the G1/S check point and G2/M check point, that ensure normal cell division. Progression through these checkpoints involves the activity of cyclin-dependent kinases and their regulatory subunits known as cyclins. In order for plants to survive, cell cycle control must be balanced with adaption to dynamic environmental conditions. In this review, we summarize recent advances in our understanding of cell cycle regulation in plants, with a focus on the molecular interactions of cell cycle machinery in the context of stress tolerance.

Phytohormones regulate plant growth from cell division to organ development. Jasmonates (JAs) are signaling molecules that have been implicated in stress-induced responses. However, they have also been shown to inhibit plant growth, but the mechanisms are not well understood. The effects of methyl jasmonate (MeJA) on leaf growth regulation were investigated in Arabidopsis (Arabidopsis thaliana) mutants altered in JA synthesis and perception, allene oxide synthase and coil-16B (for coronatine insensitive1), respectively. We show that MeJA inhibits leaf growth through the JA receptor COI1 by reducing both cell number and size. Further investigations using flow cytometry analyses allowed us to evaluate ploidy levels and to monitor cell cycle progression in leaves and cotyledons of Arabidopsis and/or Nicotiana benthamiana at different stages of development. Additionally, a novel global transcription profiling analysis involving continuous treatment with MeJA was carried out to identify the molecular players whose expression is regulated during leaf development by this hormone and COI1. The results of these studies revealed that MeJA delays the switch from the mitotic cell cycle to the endoreduplication cycle, which accompanies cell expansion, in a COI1 -dependent manner and inhibits the mitotic cycle itself, arresting cells in G1 phase prior to the S-phase transition. Significantly, we show that MeJA activates critical regulators of endoreduplication and affects the expression of key determinants of DNA replication. Our discoveries also suggest that MeJA may contribute to the maintenance of a cellular \"stand-by mode\" by keeping the expression of ribosomal genes at an elevated level. Finally, we propose a novel model for MeJA-regulated COI1 -dependent leaf growth inhibition.

Background Cyclin-dependent kinases (CDKs) critically regulate plant cell cycle transitions, including mitosis-to-endoreduplication switches essential for growth and adaptation. In Medicago truncatula , nodules form through symbiotic nitrogen fixation with rhizobia. The terminal differentiation of bacteroids within nodule cells is critical for efficient nitrogen fixation. To maintain and optimize the functionality of these differentiated symbiosomes, host nodule cells undergo repeated rounds of endoreduplication. However, which CDKs are involved in regulating endoreduplication in nodule cells to support effective symbiotic nitrogen fixation remains largely unknown. Results We identified and characterized 29 CDK genes (15 CDKs and 14 CDKLs ) classified into eight conserved subgroups. These genes displayed diverse exon/intron structures and protein motifs, with CDKA , CDKB , and CDKL subfamilies showing strong conservation with Arabidopsis thaliana . Expression analysis revealed specific downregulation of CDKB1;1 , CDKB2;2 , and CDKL13 in nodule infection to fixation zones. Protein–protein interaction (PPI) network and Gene ontology (GO) analyses demonstrated CDKB1;1 and CDKB2;2 involvement in cell cycle regulation. Overexpression of CDKB1;1 or CDKB2;2 disrupted endoreduplication and nitrogen fixation, with CDKB1;1 having the most pronounced effect, while CDKL13 appeared dispensable for symbiosis. Conclusion Our study presents the comprehensive genome-wide analysis of the CDK gene family in M. truncatula , demonstrating that the essential role of CDKB1;1 and CDKB2;2 downregulation in symbiotic nitrogen fixation and endoreduplication offers new insights into cell cycle regulation in nodules. It also identifies potential targets for improving nitrogen fixation efficiency in legumes.

METHANESULFONATE-SENSITIVE21 (MMS21) ligases having critical roles in stress protection and DNA endoreduplication/repair, respectively. To help identify their corresponding targets in Arabidopsis thaliana, we used siz1 and mms21 mutants for proteomic analyses of SUMOylated proteins enriched via an engineered SUMO1 isoform suitable for mass spectrometric studies. Through multiple data sets from seedlings grown at normal temperatures or exposed to heat stress, we identified over 1000 SUMO targets, most of which are nuclear localized. Whereas no targets could be assigned to MMS21, suggesting that it modifies only a few low abundance proteins, numerous targets could be assigned to SIZ1, including major transcription factors, coactivators/repressors, and chromatin modifiers connected to abiotic and biotic stress defense, some of which associate into multisubunit regulatory complexes. SIZ1 itself is also a target, but studies with mutants protected from SUMOylation failed to uncover a regulatory role. The catalog of SIZ1 substrates indicates that SUMOylation by this ligase provides stress protection by modifying a large array of key nuclear regulators.

Plant roots consist of a meristematic zone of mitotic cells and an elongation zone of rapidly expanding cells, in which DNA replication often occurs without cell division, a process known as endoreduplication. The duration of the cell cycle and DNA replication, as measured by 5-ethynyl-2′-deoxy-uridine (EdU) incorporation, differed between the two regions (17 h in the meristematic zone, 30 h in the elongation zone). Two distinct subnuclear patterns of EdU signals, whole and speckled, marked nuclei undergoing DNA replication at early and late S phase, respectively. The boundary region between the meristematic and elongation zones was analysed by a combination of DNA replication imaging and optical estimation of the amount of DNA in each nucleus (C-value). We found a boundary cell with 4C nuclei exhibiting the whole pattern of EdU signals. Analyses of cells in the boundary region revealed that endoreduplication precedes rapid cell elongation in roots.

The development of a new organ is the result of coordinated events of cell division and expansion, in strong interaction with each other. This study presents a dynamic model of tomato fruit development that includes cell division, endoreduplication, and expansion processes. The model is used to investigate the potential interactions among these developmental processes within the context of the neo-cellular theory. In particular, different control schemes (either cell-autonomous or organ-controlled) are tested and compared to experimental data from two contrasting genotypes. The model shows that a pure cell-autonomous control fails to reproduce the observed cell-size distribution, and that an organ-wide control is required in order to get realistic cell-size variations. The model also supports the role of endoreduplication as an important determinant of the final cell size and suggests that a direct effect of endoreduplication on cell expansion is needed in order to obtain a significant correlation between size and ploidy, as observed in real data.