BackgroundThe in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. However, current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Particularly in the gametophytic cell lineages, which are physically encapsulated in the reproductive organ structures, direct in vivo ploidy determination has been proven very challenging. Using Arabidopsis thaliana as a model, we here assess the applicability of recombinant CENH3-GFP reporters for the labeling of the cell’s chromocenters and for the monitoring of the gametophytic and somatic chromosome number in vivo.ResultsBy modulating expression of a CENH3-GFP reporter cassette using different promoters, we isolated two reporter lines that allow for a clear and highly specific labeling of centromeric chromosome regions in somatic and gametophytic cells respectively. Using polyploid plant series and reproductive mutants, we demonstrate that the pWOX2-CENH3-GFP recombinant fusion protein allows for the determination of the gametophytic chromosome number in both male and female gametophytic cells, and additionally labels centromeric regions in early embryo development. Somatic centromere labeling through p35S-CENH3-GFP shows a maximum of ten centromeric dots in young dividing tissues, reflecting the diploid chromosome number (2x = 10), and reveals a progressive decrease in GFP foci frequency throughout plant development. Moreover, using chemical and genetic induction of endomitosis, we demonstrate that CENH3-mediated chromosome labeling provides an easy and valuable tool for the detection and characterization of endomitotic polyploidization events.ConclusionsThis study demonstrates that the introgression of the pWOX2-CENH3-GFP reporter construct in Arabidopsis thaliana provides an easy and reliable methodology for determining the chromosome number in developing male and female gametes, and during early embryo development. Somatically expressed CENH3-GFP reporters, on the other hand, constitute a valuable tool to quickly determine the basic somatic ploidy level in young seedlings at the individual cell level and to detect and to quantify endomitotic polyploidization events in a non-destructive, microscopy-based manner.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0700-5) contains supplementary material, which is available to authorized users.

Chronic non-healing wounds are a major complication of diabetes, which impacts 1 in 10 people worldwide. Dying cells in the wound perpetuate the inflammation and contribute to dysregulated tissue repair 1-3 . Here, we reveal the membrane transporter Slc7a11 as a molecular 'brake' on efferocytosis, the process by which dying cells are removed, and that inhibiting Slc7a11 can accelerate wound healing. First, transcriptomics of efferocytic dendritic cells identified upregulation of several Slc7 gene family members. In further analyses, pharmacological inhibition, siRNA knockdown, or deletion of Slc7a11 enhanced dendritic cell efferocytosis. Interestingly, Slc7a11 was highly expressed in skin dendritic cells, and scRNAseq of inflamed skin showed Slc7a11 upregulation in innate immune cells. In a mouse model of excisional skin wounding, loss of Slc7a11 expression or inhibition accelerated healing dynamics and reduced apoptotic cell load in the wound. Mechanistic studies revealed a link between Slc7a11, glucose homeostasis, and diabetes. Slc7a11-deficient dendritic cells relied on glycogen store-derived aerobic glycolysis for improved efferocytosis, and transcriptomics of efferocytic Slc7a11-deficient dendritic cells identified genes linked to gluconeogenesis and diabetes. Further, Slc7a11 expression was higher in the wounds of diabetic-prone db/db mice, and targeting Slc7a11 accelerated their wound healing. The faster healing was also linked to the release of TGF- family member GDF15 from efferocytic dendritic cells. Collectively, Slc7a11 is a negative regulator of efferocytosis, and removing this brake improves wound healing, with significant implications for diabetic wound management.

SummaryCentromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non‐nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells. Here, we show that the Arabidopsis orthologue of the mammalian NUCLEAR AUTOANTIGENIC SPERM PROTEIN (NASP) and Schizosaccharomyces pombe histone chaperone Sim3 is a soluble nuclear protein that binds the histone variant CenH3 and affects its abundance at the centromeres. NASPSIM3 is co‐expressed with Arabidopsis CenH3 in dividing cells and binds directly to both the N‐terminal tail and the histone fold domain of non‐nucleosomal CenH3. Reduced NASPSIM3 expression negatively affects CenH3 deposition, identifying NASPSIM3 as a CenH3 histone chaperone.

The loading and maintenance of centromeric histone 3 (CENH3) at the centromere are critical processes ensuring appropriate kinetochore establishment and equivalent segregation of the homologous chromosomes during cell division. CENH3 loss of function is lethal, whereas mutations in the histone fold domain are tolerated and lead to chromosome instability and chromosome elimination in embryos derived from crosses with wild-type pollen. A wide range of proteins in yeast and animals have been reported to interact with CENH3. The histone fold domain-interacting proteins are potentially alternative targets for the engineering of haploid inducer lines, which may be important when CENH3 mutations are not well supported by a given crop. Here, we provide an overview of the corresponding plant orthologs or functional homologs of CENH3-interacting proteins. We also list putative CENH3 post-translational modifications that are also candidate targets for modulating chromosome stability and inheritance.

Organ- or tissue-specific ploidy level determination is often used for answering biological, molecular, genetic, or evolutionary questions in plant sciences. However, current techniques for ploidy determination either cannot provide information on single cell level, require destructive sample preparation, or are laborious and time-consuming. Here, we present a new approach developed in Arabidopsis thaliana, which is not only less labor intensive but also allows in vivo ploidy determination on single cell level. The technique is based on the incorporation of a transgenic construct, consisting of the centromere-specific protein CENH3 fused to the fluorescent reporter GFP that specifically labels centromeric regions and hence allows for an accurate visual determination of the cell's chromosome number. Moreover, by combining the construct with a gametophyte-specific promoter, the technique enables accurate chromosome quantification in all individual gametophytic cell types formed during micro- and mega-gametogenesis. As such, CENH3-based centromere visualization provides an easy and straightforward method to monitor meiotic cell division integrity, gametophytic chromosome dynamics, and reproductive ploidy stability.

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