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

Abstract

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.

Fig. 1

Modulating CENH3-GFP expression in Arabidopsis using different promoters. a-f Bright field and GFP fluorescent imaging in wild type PMCs showing autofluorescent dots at meiotic initiation a and d, prophase I b and e and tetrad stage c and f. g-r Promoter-dependent expression and localization of CENH3-GFP in somatic and reproductive organs in Arabidopsis. g pASY2-CENH3-GFP fluorescent dots in uninuclear microspores. h-j Expression of pAtPS1- and p35S-CENH3-GFP in respectively roots h and leaves i of ten-day-old seedlings. Scale bars, 10 μm. j pAtPS1-CENH3-GFP expression in mature petals. Scale bar, 50 μm. k-l GFP fluorescent imaging of pAtPS1-CENH3-GFP premeiotic cells in male sporogenesis. m-r GFP fluorescence in p35S-CENH3-GFP hypocotyls m-n, flower meristems o-p and young petals q-r. Scale bars, 10 μm, except for hypocotyls; 25 μm

Fig. 2

CENH3-GFP driven by pWOX2 labels centromeres in male gametogenesis. a-h Expression and centromere-specific localization of the pWOX2-CENH3-GFP protein during male gametophyte development; meiotic tetrad stage a, early uninuclear microspore b, mid-uninuclear microspore c, late uninuclear microspore d and e, first mitotic division with doublet CENH3-GFP dots f, binuclear microspore stage g and h. Images are processed z-stack files. Bright fluorescent dots in a and b are also observed in wild type. Scale bar, 10 μm. i Frequency distribution of male spores (n = 383) depending on the number of CENH3-GFP fluorescent dots at the late uninuclear microspore stage. Error bars represent standard deviation (of three independent analyses of at least 100 spores)

Fig. 3

Gamete-specific centromere labeling in female pWOX2-CENH3-GFP embryo sacs. a-t Bright field imaging and GFP-based assessment of pWOX2-CENH3-GFP expression and localization during female reproduction; pre-meiotic stage a and b, meiosis e and f, uninuclear megaspore i and j, binuclear embryo sac m and n and fully matured seven-celled embryo sac q and r. Arrows indicate gametophytic cells. Scale bar, 20 μm. Corresponding stages in male gametophytic development are depicted next to each ovule figure; uninuclear d and e, binuclear microspore g and h and trinuclear k, l, o, p, s and t pollen stage. Scale bar, 10 μm. Images are single snapshots and hence not all centromeric signals may be displayed

Fig. 4

Male gametophytic ploidy distribution in tetraploid Arabidopsis using pWOX2-CENH3-GFP. a-d Late uninuclear microspores from tetraploid Arabidopsis expressing pWOX2-CENH3-GFP. Images are processed z-stacks. The microspores either contain the diploid number a or an aneuploid number of centromeric GFP signals b-d. e Frequency distribution of CENH3-GFP signals in nuclei of late uninuclear microspores isolated from diploid and tetraploid Arabidopsis controls. Error bars represent standard deviation (of three independent analyses of at least 100 spores). f-h Representative images of male meiotic chromosome spreads at the start of MII in a wild type tetraploid line showing either a balanced f or an unbalanced g and h segregation of homologous chromosomes in MI. In some cases, one or more lagging chromosomes are observed h. Scale bars, 10 μm

Fig. 5

Gametophytic ploidy distribution using pWOX2-CENH3-GFP in triploid Arabidopsis. a-d Representative images of male meiotic outcome in wild type (a, unbalanced tetrad; b, polyad) and jason-2 (c, triad; d, dyad) triploid lines. e-h DAPI-stained chromosome spreads of male meiocytes at the beginning of MII in triploid Arabidopsis shows lagging chromosomes h and an unbalanced segregation of homologous chromosomes. i-l GFP fluorescence images of late uninuclear microspores isolated from a triploid pWOX2-CENH3-GFP line. Images are processed z-stacks. Scale bars, 10 μm. m-n Frequency distribution of pWOX2-CENH3-GFP signals in nuclei of late uninuclear microspores isolated from triploid Arabidopsis control lines m and a male overdose triploid jason line n. Error bars represent standard deviation (of three independent analyses of at least 100 spores). o-r Fluorescent imaging of binuclear megaspores in developing ovules of triploid pWOX2-CENH3-GFP. Scale bars, 10 μm

Fig. 6

CENH3-GFP centromere labeling allows ploidy determination during early embryogenesis. a-d Centromere-specific labeling of the recombinant pWOX2-CENH3-GFP protein in early stages of embryo and zygote development: one-celled stage a, four-celled stage b, eight-celled stage c and the late globular embryo stage d. Nuclei always show ten centromeric GFP dots. Scale bar, 20 μm

Fig. 7

Characterization of p35S-CENH3-GFP expression in Arabidopsis plant development. a-g Expression profile and centromere-specific localization of the p35S-CENH3-GFP recombinant protein in the primary root tip a and b, root elongation zone c, root differentiation zone d, the proliferating root tip e and the lateral root tip f and g. a-b Scale bars, 25 μm. c-g Scale bars, 10 μm. h-i Bright field and GFP fluorescence of a p35S-CENH3-GFP root hair. Scale bar, 20 μm. j-k Centromere-specific p35S-CENH3-GFP signals in emerging leaves of 8-day-old seedlings j and mature petals at anthesis k. Scale bars, 10 μm. l-m Frequency distribution of centromeric GFP signals in nuclei of young leaves l and petals m at different stages in development. Error bars represent standard deviation (of three independent analyses of at least 100 spores)

Fig. 8

Somatic p35S-CENH3-GFP centromere labeling detects endomitotic polyploidy. a-e Representative images of somatic nuclei expressing p35S-CENH3-GFP in young leaves of wild type a and colchicine-treated seedlings at seven days b and c and twenty days d and e after treatment. f and g Fluorescent images of p35S-CENH3-GFP localization in young developing petals in diploid wild type f and et2 g background. Note the ectopic presence of large endomitotic nuclei containing more than ten centromeric GFP signals in et2 petals. Scale bars, 10 μm