CENH3-GFP: a visual marker for gametophytic and somatic ploidy determination in Arabidopsis thaliana.

BACKGROUND: The 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. RESULTS: By 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. CONCLUSIONS: This 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.

Chromosome segregation in plant meiosis.

Faithful chromosome segregation in meiosis is essential for ploidy stability over sexual life cycles. In plants, defective chromosome segregation caused by gene mutations or other factors leads to the formation of unbalanced or unreduced gametes creating aneuploid or polyploid progeny, respectively. Accurate segregation requires the coordinated execution of conserved processes occurring throughout the two meiotic cell divisions. Synapsis and recombination ensure the establishment of chiasmata that hold homologous chromosomes together allowing their correct segregation in the first meiotic division, which is also tightly regulated by cell-cycle dependent release of cohesin and monopolar attachment of sister kinetochores to microtubules. In meiosis II, bi-orientation of sister kinetochores and proper spindle orientation correctly segregate chromosomes in four haploid cells. Checkpoint mechanisms acting at kinetochores control the accuracy of kinetochore-microtubule attachment, thus ensuring the completion of segregation. Here we review the current knowledge on the processes taking place during chromosome segregation in plant meiosis, focusing on the characterization of the molecular factors involved.

SHUGOSHINs and PATRONUS protect meiotic centromere cohesion in Arabidopsis thaliana.

In meiosis, chromosome cohesion is maintained by the cohesin complex, which is released in a two-step manner. At meiosis I, the meiosis-specific cohesin subunit Rec8 is cleaved by the protease Separase along chromosome arms, allowing homologous chromosome segregation. Next, in meiosis II, cleavage of the remaining centromere cohesin results in separation of the sister chromatids. In eukaryotes, protection of centromeric cohesion in meiosis I is mediated by SHUGOSHINs (SGOs). The Arabidopsis genome contains two SGO homologs. Here we demonstrate that Atsgo1 mutants show a premature loss of cohesion of sister chromatid centromeres at anaphase I and that AtSGO2 partially rescues this loss of cohesion. In addition to SGOs, we characterize PATRONUS which is specifically required for the maintenance of cohesion of sister chromatid centromeres in meiosis II. In contrast to the Atsgo1 Atsgo2 double mutant, patronus T-DNA insertion mutants only display loss of sister chromatid cohesion after meiosis I, and additionally show disorganized spindles, resulting in defects in chromosome segregation in meiosis. This leads to reduced fertility and aneuploid offspring. Furthermore, we detect aneuploidy in sporophytic tissue, indicating a role for PATRONUS in chromosome segregation in somatic cells. Thus, ploidy stability is preserved in Arabidopsis by PATRONUS during both meiosis and mitosis.

Volume-based pollen size analysis: an advanced method to assess somatic and gametophytic ploidy in flowering plants.

Pollen size is often used as a biological parameter to estimate the ploidy and viability of mature pollen grains. In general, pollen size quantification is performed one- or two-dimensionally using image-based diameter measurements. As these approaches are elaborate and time consuming, alternative approaches that enable a quick, reliable analysis of pollen size are highly relevant for plant research. In this study, we present the volume-based particle size analysis technique as an alternative method to characterize mature pollen. Based on a comparative assay using different plant species (including tomato, oilseed rape, kiwifruit, clover, among others), we found that volume-based pollen size measurements are not biased by the pollen shape or position and substantially reduce non-biological variation, allowing a more accurate determination of the actual pollen size. As such, volume-based particle size techniques have a strong discriminative power in detecting pollen size differences caused by alterations in the gametophytic ploidy level and therefore allow for a quick and reliable estimation of the somatic ploidy level. Based on observations in Arabidopsis thaliana gametophytic mutants and differentially reproducing Boechera polyantha lines, we additionally found that volume-based pollen size analysis provides quantitative and qualitative data about alterations in male sporogenesis, including aneuploid and diploid gamete formation. Volume-based pollen size analysis therefore not only provides a quick and easy methodology to determine the somatic ploidy level of flowering plants, but can also be used to determine the mode of reproduction and to quantify the level of diplogamete formation.