Point mutation impairs centromeric CENH3 loading and induces haploid plants

Abstract

The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called "CENP-A") is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923-937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest.

Keywords: CENH3 loading; CENH3 mutant; chromosome elimination; haploid induction; plant breeding.

Fig. 1.

Centromeres of barley TILLING line 4528 lost βCENH3. Chromosomes of wild-type and homozygous TILLING line 4528 after immunostaining with antibodies specific for αCENH3 (green) and βCENH3 (red). Note the absence of βCENH3-specific signals in line 4528.

Fig. S1.

Transcription level of αCENH3 and βCENH3 in wild-type (cv Barke) and TILLING line 4528 with mutated βCENH3. The relative expression level of αCENH3 and βCENH3 was measured in different tissues of barley using specific primers (Table S1). cDNA was prepared from total RNA, and gene expression levels were normalized to GAPDH. Each value represents the mean ± SD (n = 3). 7, 7 d after pollination; 14, 14 d after pollination.

Fig. S2.

The phenotypes of wild-type and homozygous TILLING line 4528 barley plants are similar. (A) Relative proportion of 2C, 4C, 8C, and 16C leaf nuclei determined by flow cytometry. (B) The cycle values indicating the mean numbers of endoreduplication cycles per nucleus (32) in wild-type 1–3 and in TILLING line 4528 lines 1–3 plants. (C) Growth habit of TILLING line 4528 (Left) and wild-type (Right) plants.

Fig. S3.

The CENH3 mutation is located in an evolutionarily highly conserved C-terminal CENP-A targeting domain (CATD) defined by loop 1 and helix 2 of the histone fold domain. Shown are multiple alignments of partial sequences of the CENH3 proteins from different monocot (Hordeum vulgare_βCENH3: JF419329; Hordeum vulgare_αCENH3: JF419328; Hordeum bolbusom_βCENH3: JF419330; Hordeum bolbusom_αCENH3: GU245882; Allium cepa: BAL45432; Avena sativa: AB981584.1; Oryza sativa: AY438639; Saccharum officinarum: CA127217; Sorghum bicolor: XM_002441245.1; Triticum aestivum: JF969286.1; and Zea mays: AF519807) and eudicot (Arabidopsis thaliana: AF465800; Beta vulgaris (this study); Brassica juncea: BAF49728; Brassica napus: ACZ04984.1; Brassica rapa: NM_001302028.1; Daucus carota: KJ201903; Glycine max: FK014964; Gossypium arboreum: KP177465; Gossypium hirsutum: KP177475.1; Gossypium raimondii: KP177464.1; Lepidium virginicum: BAF49732; Nicotiana tabacum: AB366153.1; Raphanus sativus: BAF49733.1; Solanum lycopersicum: XM_010328624-1; and Solanum tuberosum: XM_006339625.1) plant species and nonplant species (Drosophila melanogaster: AY126930; and Homo sapiens: AAP36900). The single point mutation in barley βCENH3 is indicated by the black arrow.

Fig. 2.

Characterization of the CENH3 point mutation in A. thaliana and B. vulgare. (A) Reduced centromere targeting of L130-mutated CENH3 in A. thaliana. (Upper) Quantification of centromere colocalization patterns (complete, partial, and no) in flower bud nuclei of wild-type A. thaliana (502 nuclei from three plants), cenh3 L130I (543 nuclei from four plants), and cenh3 L130F (1,105 nuclei from seven plants). (Lower) Representative double immunostaining patterns. Construct and endogenous wild-type CENH3 were detected with anti-GFP and anti-CENH3 antibodies, respectively. (B) Reduced centromere targeting of L106-mutated CENH3 in B. vulgaris. (Upper) Quantification of centromere colocalization patterns (complete, partial, and no) in callus and leaves of plants transformed with RFP reporter of wild-type CENH3 (230 nuclei from five plants) and Bvcenh3 L106I (160 nuclei from two plants) or Bvcenh3 L106F (350 nuclei from five plants) constructs. (Lower) Representative centromere patterns. (C and D, Upper) Distribution of CENH3 in Atcenh3 L130F-complemented cenh3.1-null mutants. Quantification of centromere colocalization patterns (strong, weak, or dispersed) in leaf nuclei of wild-type (1,071 nuclei from three plants), cenh3 L130F-1 (2,666 nuclei from six plants), cenh3 L130F-2 (1,157 nuclei from three plants), and cenh3 L130F-3 (1,656 nuclei from four plants) (C) and in sperm nuclei of wild-type (1,121 nuclei from three plants), cenh3 L130F-1 (1,918 nuclei from five plants), cenh3 L130F-2 (1,321 nuclei from three plants), and cenh3 L130F-3 (1,201 nuclei from three plants). (C and D, Lower) Representative anti-CENH3 distribution patterns. Double asterisks indicate 1% significance versus CENH3 wild type; single asterisks indicate 5% significance versus CENH3 wild type. (E) Flow histograms of diploid and haploid A. thaliana plants together with representative nuclei after FISH using an A. thaliana centromere-specific probe (in red).

Fig. S4.

Analysis of Atcenh3 L130F A. thaliana lines. (A) Determination of the Atcenh3 L130F transgene copy number by Southern blot. DNA blot [T2 plants of L130F-1 (two plants), L130F-2 (three plants), and L130F-3 (three plants)] were probed with a labeled hygromycin resistance gene. (B) Growth habit of wild-type and Atcenh3 L130F-1. Insets show the corresponding flower phenotype. (C) Analysis of meiosis, pollen quality, seed phenotype, germination frequency, and ploidy. The asterisk indicates the occurrence of lagging chromosomes or micronuclei.

Fig. S5.

Haploid plants have only a wild-type CENH3 allele. Genotyping by dCAPS PCR. The Atcenh3-1 L130F mutant allele is not cut (215 bp); the CENH3 wild allele is cut (191 and 24 bp). cenh3-1/cenh3-1, complemented plant by genomic construct of cenh3 L130F; CENH3/cenh3-1, hybrid between Atcenh3 L130F and a wild-type plant. CENH3/CENH3, wild-type plant.

Fig. S6.

Scanning electron micrographs showing phenotypes of F1 seeds derived from the Atcenh3 L130F1 × wild-type A. thaliana cross.

Fig. S7.

Relative amount of CENH3 in different Atcenh3 L130F lines. The relative amount of CENH3 (CENH3/histone H3 signal intensity ratio) was determined by quantitative Western blot analysis with A. thaliana CENH3-specific antibodies and histone H3-specific antibodies (n = 3 plants of each line). Error bars indicate the 95% confidence interval. Double asterisks indicate 1% significance versus wild type.

Fig. 3.

Model explaining the uniparental chromosome elimination in haploid inducer CENH3 × wild-type CENH3 intraspecific hybrid embryos (Left) compared with embryos originating from A. thaliana wild-type crossings (Right). (A) Diploid homozygous haploid inducer and wild-type CENH3 parents produce haploid gametes during meiosis. (B) Egg cells derived from the haploid inducer likely contain either less CENH3 than eggs derived from wild type or a reduced amount of an unknown CENH3-transgeneration required signature. (C) After fertilization, the paternal wild-type CENH3 is actively removed from the zygote nucleus. (D) Centromeric reloading of wild-type CENH3 in the zygote occurs at the 16-nuclei stage of endosperm development in A. thaliana. (E and F) In embryos undergoing haploidization, centromeric reloading of the maternal chromosomes is impaired or delayed causing (i) lagging chromosomes and (ii) subsequent micronuclei formation because of centromere inactivity (E). Subsequently, micronucleated haploid-inducer chromosomes will degrade, and a haploid embryo will develop (F). Embryos contain paternal-derived chromosomes in the background of maternal-derived cytoplasm.