Characterization of Centromeric Histone H3 (CENH3) Variants in Cultivated and Wild Carrots (<i>Daucus</i> sp.)

<div><p>In eukaryotes, centromeres are the assembly sites for the kinetochore, a multi-protein complex to which spindle microtubules are attached at mitosis and meiosis, thereby ensuring segregation of chromosomes during cell division. They are specified by incorporation of CENH3, a centromere specific histone H3 variant which replaces canonical histone H3 in the nucleosomes of functional centromeres. To lay a first foundation of a putative alternative haploidization strategy based on centromere-mediated genome elimination in cultivated carrots, in the presented research we aimed at the identification and cloning of functional CENH3 genes in <i>Daucus carota</i> and three distantly related wild species of genus <i>Daucus</i> varying in basic chromosome numbers. Based on mining the carrot transcriptome followed by a subsequent PCR-based cloning, homologous coding sequences for CENH3s of the four <i>Daucus</i> species were identified. The ORFs of the CENH3 variants were very similar, and an amino acid sequence length of 146 aa was found in three out of the four species. Comparison of <i>Daucus</i> CENH3 amino acid sequences with those of other plant CENH3s as well as their phylogenetic arrangement among other dicot CENH3s suggest that the identified genes are authentic CENH3 homologs. To verify the location of the CENH3 protein in the kinetochore regions of the <i>Daucus</i> chromosomes, a polyclonal antibody based on a peptide corresponding to the N-terminus of <i>DcCENH3</i> was developed and used for anti-CENH3 immunostaining of mitotic root cells. The chromosomal location of CENH3 proteins in the centromere regions of the chromosomes could be confirmed. For genetic localization of the CENH3 gene in the carrot genome, a previously constructed linkage map for carrot was used for mapping a CENH3-specific simple sequence repeat (SSR) marker, and the CENH3 locus was mapped on the carrot chromosome 9.</p></div

RT-PCR-based transcriptional analysis of CENH3 in <i>D. carota</i> (Dcar), <i>D. glochidiatus</i> (Dglo), <i>D. pusillus</i> (Dpus) and <i>D. muricatus</i> (Dmur) with gene-specific primer pairs designed for <i>D. carota</i> CENH3 (DcEXP) and CENH3s of <i>D. pusillus</i>/<i>D. glochidiatus</i> (DpgEXP).

<p>For details, see text, and for position of primers, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098504#pone.0098504.s001" target="_blank">Figure S1</a>. For the reference gene <i>β-actin</i> the primer pair DcACT was used. Positive control is genomic DNA of <i>D. carota, D. glochidiatus</i> and <i>D. pusillus,</i> and negative control is water (W). Size standard (M) is the Gene Ruler DNA ladder Mix (Thermo Fisher Scientific).</p

Double-immunostaining of root-tip cells of carrot (<i>D</i>. <i>carota</i>) at different stages of mitosis with antibodies against carrot CENH3 (in red) and α-tubulin (in green).

<p>Chromosomes are counterstained with DAPI (in blue). (A–D) prophase, (E–H) metaphase, (I–L) anaphase, (M–P) telophase. Scale bar 10 µm.</p

Immunostaining of <i>Daucus</i> root tip cells using anti-<i>DcCENH3</i> antibody.

<p>(<b>A</b>–<b>C</b>) <i>D. carota</i> (2n = 2x = 18) metaphase chromosomes, (<b>D</b>–<b>F</b>) <i>D. glochidiatus</i> (2n = 4x = 44) metaphase chromosomes, (<b>G</b>, <b>H</b>) interphase nuclei of <i>D. carota</i>. A, D and also G are DAPI-stained chromosomes, B, E and also H are CENH3 immunosignals, C and F are merged images. Scale bar 5 µm.</p

Phylogenetic tree of the deduced <i>Daucus</i> CENH3 proteins (printed in bold letters) and a selection of plant CENH3 proteins representing monocot (Alliaceae, Poaceae) and various dicot plant families including Leguminosae and Brassicaceae.

<p>Canonical histone H3 of <i>A. thaliana</i> was used as an outgroup. For each amino acid sequence, the NCBI accession number is indicated in parentheses. Multiple sequence alignment was performed by ClustalW using the Lasergene (DNASTAR) software package. A phylogenetic tree was constructed using the Kimura distance formula to calculate distance values and bootstrap analysis (10,000 replicates). Numbers indicate bootstrap replication, and branch length is scaled below the tree indicating the number of amino acid substitutions per 100 amino acids.</p

Genetic map of the carrot chromosome 9 (corresponding to linkage group 7) with the calculated position of the <i>DcCENH3</i> gene mapped through the DCEN-SSR marker.

<p>Scale: centiMorgan (cM).</p

Multiple sequence alignment of the deduced <i>Daucus</i> CENH3 proteins and comparison with CENH3 sequences from <i>Nicotiana tabacum</i> (GenBank accession number BAH03515) and <i>Vitis vinifera</i> (XP_002281073) showing the highest similarity to <i>Daucus</i> CENH3s after multiple alignment of various plant CENH3 proteins (see <b>Figure 2</b>).

<p>Sequences were compared by ClustalW (Lasergene). The putative centromere targeting domain (CATD) spanning loop 1 and α-2 helix is marked by a crossbar. The position used for construction of a peptide antibody against <i>DcCENH3</i> is boxed.</p

dataset for all 114 strains

The whole dataset is available for download(114 strains). Note that the observed 106 unique haplotypes (i.e. the clone-corrected dataset used for all the analyses; see the Methods section) are labeled with a "0". Excluded clones are labeled with a "1"

Bioactive C₁₇-Polyacetylenes in Carrots (Daucus carota L.): Current Knowledge and Future Perspectives

C₁₇-polyacetylenes (PAs) are a prominent group of oxylipins and are primarily produced by plants of the families Apiaceae, Araliaceae, and Asteraceae, respectively. Recent studies on the biological activity of polyacetylenes have indicated their potential to improve human health due to anticancer, antifungal, antibacterial, anti-inflammatory, and serotogenic effects. These findings suggest targeting vegetables with elevated levels of bisacetylenic oxylipins, such as falcarinol, by breeding studies. Due to the abundant availability, high diversity of cultivars, worldwide experience, and high agricultural yields, in particular, carrot (Daucus carota L.) genotypes are a very promising target vegetable. This paper provides a review on falcarinol-type C₁₇-polyacetylenes in carrots and a perspective on their potential as a future contributor to improving human health and well-being