Heat stress reveals a specialized variant of the pachytene checkpoint in meiosis of Arabidopsis thaliana

Abstract

Plant growth and fertility strongly depend on environmental conditions such as temperature. Remarkably, temperature also influences meiotic recombination and thus, the current climate change will affect the genetic make-up of plants. To better understand the effects of temperature on meiosis, we followed male meiocytes in Arabidopsis thaliana by live cell imaging under three temperature regimes: at 21°C; at heat shock conditions of 30°C and 34°C; after an acclimatization phase of 1 week at 30°C. This work led to a cytological framework of meiotic progression at elevated temperature. We determined that an increase from 21°C to 30°C speeds up meiosis with specific phases being more amenable to heat than others. An acclimatization phase often moderated this effect. A sudden increase to 34°C promoted a faster progression of early prophase compared to 21°C. However, the phase in which cross-overs mature was prolonged at 34°C. Since mutants involved in the recombination pathway largely did not show the extension of this phase at 34°C, we conclude that the delay is recombination-dependent. Further analysis also revealed the involvement of the ATAXIA TELANGIECTASIA MUTATED kinase in this prolongation, indicating the existence of a pachytene checkpoint in plants, yet in a specialized form.

Figure 1

Localization of CDKA;1 in male meiocytes under control and stress conditions. A–C, CDKA;1-mVenus (first row; white) and TagRFP-TUA5 (second row; magenta) localization under the control conditions of 21°C (A), heat stress of 30°C (B), or 34°C (C) at different meiotic stages: G2-early leptotene (Column 1), late leptotene-early pachytene (Column 2), pachytene-diakinesis (Column 3), metaphase I-interkinesis (Column 4), and metaphase II-telophase II (Column 5). Red arrowheads highlight cells with CDKA;1 localization at SGs. Scale bar, 10 µm. D, Quantification of CDKA;1 SG formation on the cellular level per stage in percent; white bar, cells without SGs; gray bar, cells with at least one SG. The absolute sample size is given in the corresponding bar.

Figure 2

Duration of meiotic phases based on MT array states. Confocal images of MT array states (A–F) and the corresponding predicted median times (in minutes) with 95% CIs in control (21°C) and heat conditions (HS30°C, HS34°C, and LT30°C) (A′—F′); (A, A′; orange) MT array state 2-3-4, late leptotene-early pachytene; (B, B′; green) MT array state 5-6, pachytene-diakinesis; (C, C′; light blue) MT array state 7-8-9, metaphase I-anaphase I; (D, D′; purple) MT array state 10-11, telophase I-interkinesis; (E, E′; dark blue) MT array state 12-13, metaphase II-anaphase II; (F, F’; gray) MT array state 14, telophase II. Scale bar, 10 µm. G, Predicted median time (in minutes) of MT array states 2–13 with the 95% CI in control (21°C) and heat conditions (HS30°C, HS34°C, and LT30°C) (yellow).

Figure 3

MT array in the WT in control and heat stress conditions. A–D, Confocal images of meiocytes expressing TagRFP-TUA5 (magenta) at MT array state 6 in control conditions of 21°C (A), heat shock conditions of HS30°C (B), LT30°C (C), and HS34°C (D). A′–D′, Pixel intensity plot of a section crossing through the middle of the cell (distance in um) in MT array state 6 in 21°C (A′, green, n = 14), HS30°C (B′, yellow, n = 17), LT30°C (C′, blue, n = 31) and HS34°C (D′, brown, n = 32), section lines also highlighted in (A–D). E, Confocal images of meiocytes expressing TagRFP-TUA5 (magenta) at MT array state 8-9 at 21°C, HS30°C, LT30°C, and HS34°C. Scale bar, 10 µm.

Figure 4

Localization of the synaptonemal complex elements ASY1 and ZYP1b upon heat stress. Confocal images of the nucleus of meiocytes at 21°C (A), HS30°C (B), and HS34°C (C) of SC elements ASY1-RFP (magenta, first row) and ZYP1b-GFP (green, second row) separately and merged (third row) at zygotene (Columns 1–3) and pachytene (Columns 4 and 5). Scale bar, 10 µm.

Figure 5

Effect of early and late heat shock on the duration of MT array state 5-6. Predicted median time (in minutes) with 95% CIs of pachytene-diakinesis (MT array state 5-6; green) at 21°C, early HS30°C versus late HS30°C and early HS34°C versus late HS34°C. 21°C, early HS30°C, and early HS34°C, as shown in Figure  2B’.

Figure 6

Duration of prophase in the recombination mutants spo11-1, dmc1, msh4, and atm at 21°C and HS34°C. A and B, Predicted median times (in minutes) with 95% CI of (A) late leptotene-early pachytene (MT array state 2-3-4; orange) and (B) pachytene-diakinesis (MT array state 5-6; green) in the WT (as shown in Figure  2, A′ and B′) and recombination mutants spo11-1, dmc1, msh4, and atm at 21°C and HS34°C.