Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids

Abstract

Uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanism underlying selective elimination of the paternal chromosomes during the early development of Hordeum vulgare × Hordeum bulbosum embryos. The following conclusions regarding the role of the centromere-specific histone H3 variant (CENH3) in the process of chromosome elimination were drawn: (i) centromere inactivity of H. bulbosum chromosomes triggers the mitosis-dependent process of uniparental chromosome elimination in unstable H. vulgare × H. bulbosum hybrids; (ii) centromeric loss of CENH3 protein rather than uniparental silencing of CENH3 genes causes centromere inactivity; (iii) in stable species combinations, cross-species incorporation of CENH3 occurs despite centromere-sequence differences, and not all CENH3 variants get incorporated into centromeres if multiple CENH3s are present in species combinations; and (iv) diploid barley species encode two CENH3 variants, the proteins of which are intermingled within centromeres throughout mitosis and meiosis.

Fig. 1.

Anaphase chromosome segregation behavior of normally segregating (A) and lagging (B) H. bulbosum chromosomes in an unstable H. vulgare × H. bulbosum hybrid embryo. Chromosomes of H. bulbosum (green) were identified by genomic in situ hybridization using labeled genomic DNA of H. bulbosum. Chromosomes of H. vulgare are shown in blue. Note the lagging chromosomes of H. bulbosum in B. (Scale bar: 10 μm.)

Fig. 2.

Anaphase chromosomes of an unstable (A) and stable (B) H. vulgare × H. bulbosum hybrid embryo after immunostaining with anti-grass CENH3 and anti–α-tubulin. The centromeres of lagging chromosomes (arrowheads) are CENH3-negative. (Scale bar: 10 μm.)

Fig. 3.

Interphase nucleus of an unstable H. vulgare × H. bulbosum hybrid embryo (A) after immunostaining with anti-grass CENH3 (B), genomic in situ hybridization with H. bulbosum DNA (C, red), and in situ hybridization with the Hordeum centromere-specific probe BAC7 (D). GISH, genomic in situ hybridization. (E) Only approximately 7 of the 14 more or less equally sized centromeric FISH signal clusters are overlapping with the position of strong CENH3 signals. Hence, interphase centromeres of H. bulbosum carry less CENH3 protein. BAC7-positive centromeres without CENH3-signals are shown (arrows). (Scale bar: 10 μm.)

Fig. 4.

Characterization of micronuclei of unstable H. vulgare × H. bulbosum hybrid embryos. Micronuclei are H. bulbosum-positive after genomic in situ hybridization (A) CENH3-negative (B) and RNA polymerase II-negative (C) after immunostaining but enriched in H3K9me2-specific heterochromatin-specific markers (D). Arrowheads indicate micronuclei. (Scale bar: 10 μm.)

Fig. 5.

Western blot analysis demonstrating the specificity of anti-HvαCENH3-, anti-HvβCENH3-, and anti-grass CENH3-specific antibodies on nuclear (A) and in vitro translated HvαCENH3 and HvβCENH3 proteins (B).

Fig. 6.

Distribution of αCENH3 and βCENH3 in mitotic (A) and meiotic (B) metaphase chromosomes and interphase nuclei of H. vulgare (C) as demonstrated by immunostaining. An overlap of αCENH3 and βCENH3 signals was found for centromeres as well as for (D) up to 12-fold artificially extended centromeres. (Scale bars: 10 μm.)

Fig. 7.

Proposed model of how the mitosis-dependent process of uniparental chromosome elimination operates in H. vulgare × H. bulbosum hybrid embryos. (A) After fertilization of the H. vulgare egg by the H. bulbosum sperm, all parental CENH3s are transcriptionally active. (B) Translation of HvCENH3s occurs; whether translation of HbCENH3s occurs is not known. (C) Loading of CENH3 (red) into the centromeres of H. vulgare but not of H. bulbosum occurs. As a result of cell cycle asynchrony of the two parental genomes, CENH3 incorporation probably only occurs in the centromeres of H. vulgare during G2. A reduced temperature during early embryogenesis promotes normal centromere activity of both parental genomes. (D) As a result of centromere inactivity in unstable hybrids, anaphase chromosomes of H. bulbosum lag and subsequently form micronuclei. (E) Micronucleated H. bulbosum chromatin finally degrades, and a haploid H. vulgare embryo will develop.

Fig. P1.

Potential mechanism for the mitosis-dependent process of uniparental chromosome elimination in H. vulgare × H. bulbosum hybrid embryos. (A) After fertilization of the H. vulgare egg by the H. bulbosum sperm, all parental CENH3s are transcriptionally active. (B) Translation of HvCENH3s occurs; however, whether translation of HbCENH3s occurs is not known. (C) Loading of CENH3 (red) into the centromeres of H. vulgare but not of H. bulbosum occurs. As a result of cell cycle asynchrony of the two parental genomes, CENH3 is likely incorporated into the centromeres of H. vulgare during the G2 phase. A reduced temperature during early embryogenesis promotes the normal centromere activity of both parental genomes. (D) As a result of centromere inactivity in unstable hybrids, anaphase chromosomes of H. bulbosum lag and subsequently form micronuclei. (E) Micronucleated H. bulbosum chromatin finally degrades, and a haploid H. vulgare embryo develops.