High temperatures alter cross-over distribution and induce male meiotic restitution in Arabidopsis thaliana

Abstract

Plant fertility is highly sensitive to elevated temperature. Here, we report that hot spells induce the formation of dyads and triads by disrupting the biogenesis or stability of the radial microtubule arrays (RMAs) at telophase II. Heat-induced meiotic restitution in Arabidopsis is predominantly SDR-type (Second Division Restitution) indicating specific interference with RMAs formed between separated sister chromatids. In addition, elevated temperatures caused distinct deviations in cross-over formation in male meiosis. Synapsis at pachytene was impaired and the obligate cross-over per chromosome was discarded, resulting in partial univalency in meiosis I (MI). At diakinesis, interconnections between non-homologous chromosomes tied separate bivalents together, suggesting heat induces ectopic events of non-homologous recombination. Summarized, heat interferes with male meiotic cross-over designation and cell wall formation, providing a mechanistic basis for plant karyotype change and genome evolution under high temperature conditions.

Fig. 1. Heat induces defects in Arabidopsis male meiotic cell division.

Representative images of lactopropionic orcein-stained tetrad-stage male meiocytes (a–d) and early-, mid-, and late-stage microspores (e–p) of Arabidopsis qrt1–2^−/− plants grown under normal temperature conditions (18–20 °C) (a, e, i, m) and at different time periods following heat treatment (24 h at 30–32 °C) (b–d, f–h, j–l, n–p). Hpt means hours post treatment. Scale bar, 10 μm.

Fig. 2. Arabidopsis thaliana male meiosis exposed to heat (30–32 °C) yields dyads, triads, and polyads.

Quantitative analysis of the different types of male meiotic figures produced by Arabidopsis thaliana 2× Col-0 male sporogenesis exposed to varying periods of moderate heat stress (30–32 °C). Values represent the mean of >500 male meiotic products isolated from at least three different plants. The total number of male meiotic products analyzed for each specific treatment is indicated by value n. Error bars represent standard deviation values.

Fig. 3. Arabidopsis male meiosis exposed to heat shows defects in MII cell wall formation.

a–h Anilin blue staining of tetrad-stage male meiocytes of diploid Arabidopsis Col-0 plants under normal temperature conditions (18 °C, a) and upon exposure to heat (24 h at 30–32 °C, b–h). i–p Anilin blue staining of early-stage microspores originating from heat-stressed Arabidopsis PMCs as viewed via bright-field (i–l) and fluorescent microscopy (m–p). Scale bar, 10 μm.

Fig. 4. Heat-induced defects in MII cell wall formation are attributed to alterations in RMA biogenesis.

Tubulin α immunostaining of Arabidopsis Col-0 male meiocytes at telophase II under normal temperatures (18–20 °C, a–c) and upon heat stress (24 h at 30–32 °C, d–f). Arrows indicate for the occurrence of two or more nuclei at telophase II that lack the presence of an internuclear phragmoplast-like structure. Scale bar, 10 μm.

Fig. 5. Heat-induced bi- and polynuclear spores show nuclear fusion before PMI to yield di- and polyploid pollen grains.

a–o In vivo fluorescent analysis of nuclear dynamics and centromere number during male gametogenesis in spores resulting from Arabidopsis male meiocytes that experienced normal temperatures (18 °C, a, d, g, j, m) and high-temperature stress (24 h at 30–32 °C, b–c, e–f, h–i, k–l, n–o) using the pWOX2-CENH3-GFP reporter. Scale bar, 10 μm. p–w In vivo analysis of the number and size of sperm nuclei in mature pollen originating from control (18 °C, p, t) and heat-stressed (24 h at 30–32 °C, 7 dpt, q–s and u–w) Arabidopsis male meiocytes using the pMGH3-H2B-GFP reporter. Scale bar, 20 μm.

Fig. 6. Heat-induced meiotic restitution in Arabidopsis predominantly yields SDR-type 2n pollen.

a Principle and examples of quantitative analysis of FDR/SDR-type male meiotic restitution in Arabidopsis qrt1–2^−/− plants using segregation analysis of centromere-linked FTL reporters in pollen dyads and triads. Representative bright-field and fluorescent images of qrt1–2^−/− tetrad, dyad, and triad configurations at Arabidopsis anthesis displaying segregation of two centromere-linked FTL markers, i.e., FTL_3332 (YFP) and FTL_2536 (dsRed), in the corresponding haploid and diploid pollen grains. Scale bar, 20 μm. b Frequency of heat-induced qrt1–2^−/− dyads and triads showing SDR-type segregation of different single FTL reporters in function of their physical distance from the centromere. The closer the FTL reporter is located to the centromere, the higher the frequency of dyads and triads with co-segregation of the corresponding FTL marker, indicating for SDR-type meiotic restitution. Corresponding quantitative data are provided in Supplementary Table 1. c Physical position of the different FTL markers on chromosomes 3 and 5 used for FDR/SDR assay together with indications of the corresponding interval. d Representative images of tetrad-stage male meiocytes in haploid Arabidopsis Col-0 plants under normal conditions (18 °C, a, b) and upon exposure to heat (24 h at 30–32 °C, c, d). Scale bar, 10 μm. e Comparative analysis of the frequency of different types of male meiotic products formed in haploid Arabidopsis plants grown under standard temperature conditions (18 °C) or upon exposure to heat (24 h at 30–32 °C). Statistical differences in the frequency of a specific meiotic product between control conditions and heat stress were tested using one-way ANOVA (α = 0.05) and are indicated by asterisks. For each treatment, at least three independent plants were analyzed. The total number of male meiotic products assessed in each treatment is indicated by value n.

Fig. 7. Partial asynapsis and anaphase I chromosome bridges in Arabidopsis male meiosis exposed to a short period of heat stress (6 h at 30–32 °C).

DAPI-stained chromosome spreads of Arabidopsis male meiocytes under standard temperature (18 °C, a–c) and upon exposure to a short period of heat stress (6 h, 30–32 °C, d–o) at three distinct phases in meiosis I, namely pachytene, diakinesis, and anaphase I. Arrows indicate for chromosomal regions that are not synapsed (d, g, j, m) or for ectopic DNA interconnections between nonhomologous chromosomes in diakinesis (e, h, k, n). The asterisk indicates for the occurrence of a single chromosome fragment at pachytene. Scale bar, 20 μm.

Fig. 8. Partial asynapsis, univalents, and anaphase I bridges in Arabidopsis male meiosis exposed to a prolonged period of heat stress (24 h at 30–32 °C).

DAPI-stained chromosome spreads of Arabidopsis male meiocytes under standard temperature (18 °C, a–f) and upon exposure to a prolonged period of heat stress (24 h at 30–32 °C, g–x) at different stages in MI and MII. Image insets are magnifications of chromosomal regions that show lack of synapsis (g, m, s). Arrows indicate for the occurrence of univalents in diakinesis (h, n), aberrant chromosome positioning in metaphase II (k, q), and occurrence of extra nuclear domains in meiotic tetrads (r, x). The asterisk indicates ectopic interconnection between bivalent chromosome structures at diakinesis. Scale bar, 10 μm.

Fig. 9. Heat-induced anaphase I chromosome bridges are DSB-dependent.

DAPI-stained chromosome spreads of male meiocytes of wild-type (a, c, e) and atspo11–1–3^−/− (b, d, f) Arabidopsis plants under heat stress (24 h at 30–32 °C) at three distinct MI phases, namely pachytene, diakinesis, and anaphase I. Scale bar, 10 µm.

Fig. 10. Heat stress induces ectopic occurrence of nonhomologous recombination in male meiosis of haploid Arabidopsis.

DAPI-stained chromosome spreads of male meiocytes of wild-type haploid Arabidopsis Col-0 plants under standard temperature conditions (18–20 °C, a–d) and upon heat stress (24 h at 30–32 °C, e–l). Arrows indicate for ectopic physical connections between nonhomologous chromosomes in metaphase I. Scale bar,10 µm.