Dividing cortical cells initiating Medicago nodule primordium formation are tetraploid
(A) Confocal images of whole-mount WT transgenic roots expressing mCitrine-CENH3 under the control of native (M.t. pCENH3; left and middle panels) or constitutive promoters (pLjUbi; right panel). Simultaneous expression of H3.3-mCherry enables the recognition of condensed (star) and segregating chromosomes (middle panel). mCitrine-CENH3 labels the centromeric region of individual chromosomes (filled arrowheads). The number of CENH3-labeled foci determined across image stacks is indicated in yellow. Images are maximum intensity projections and show merged fluorescent channels (mCitrine: Green Fire Blue; mCherry and Calcofluor white cell-wall staining: grayscale). In the Green Fire Blue colour scheme, blue or yellow indicate low or high fluorescence levels, respectively (min. to max. = 1-140 in the middle panel). Scale bars: left and right panels = 10 μm; middle panel = 2 μm. (B) Quantification of the number of centromeric signals in transgenic root tips expressing mCitrine-CENH3 under native (pCENH3) or constitutive (pUbi) promoters (n = 55 nuclei). All data points are shown and are from 2 (pUbi) to 3 (pCENH3) independent experiments with 7 composite plants analyzed per construct. Horizontal bars indicate sample means with 95% confidence interval. Differences were statistically significant (p-value = 0.0006) using a Mann-Whitney test. (C) Schematic representation of an early nodule primordium where anticlinal cell divisions occurred in the pericycle (P; dark violet) and cortical cells (C2 to C5; light violet). Rhizobia inside the infection thread are depicted in red. Ep: epidermis. En: endodermis. (D) Maximum intensity projections of an early nodule primodium at the developmental stage represented in (C), formed in a WT root expressing the pLjUbi::mCitrine-CENH3 / pH3.3::H3.3-mCherry construct at 7 dpi with mCherry-producing S. meliloti. Dotted-line ROIs indicate the contours of divided cells in the inner (C4) and middle (C3) cortical layers. The number of CENH3-labeled foci, determined across image stacks and indicated in yellow are: 11 and 16 in undivided, infected cells (IC) from the outer cortical layers (C1; left panel and C2, right panel) and 25 to 28 in divided, uninfected cells from the C3 (filled arrowhead; left panel) and C4 layers (right panel). Images show merged fluorescent channels (mCitrine: Green Fire Blue; mCherry and Calcofluor white cell-wall staining: grayscale). In the Green Fire Blue colour scheme, blue or yellow indicate low or high fluorescence levels, respectively. Scale bars: 20 μm. (E) Quantification of the number of centromeric signals in early nodule primordia at the developmental stage depicted in (C), formed in inoculated WT roots (7 to 12 dpi) constitutively expressing mCitrine-CENH3. The number (n) of analyzed nuclei in each cell layer is indicated on the top. E: endodermis. P: pericycle. All data points are shown with black or violet symbols indicating undivided or divided cells, respectively. NI: non-infected cell (discs). I: infected cell (triangles). Horizontal dotted lines are positioned at y = 16 and y = 32, corresponding to diploid (2n = 16) or tetraploid (4n = 32) cellular states in Medicago. Data are from 2 independent experiments with 7 nodule primordia from 5 composite plants analyzed.