The recruitment of the A-type cyclin TAM to stress granules is crucial for meiotic fidelity under heat

Abstract

Stress granules (SG) are biomolecular condensates that represent an adaptive response of cells to various stresses, including heat. However, the cell type-specific function and relevance of SG formation, especially during reproductive development, are largely not understood. Here, we show that the meiotic A-type cyclin TARDY ASYNCHRONOUS MEIOSIS (TAM) is recruited to SGs in male meiocytes of Arabidopsis after exposure to heat. We find that the amino terminus of TAM is necessary and sufficient for the localization of proteins to meiotic SGs. Swapping the amino terminus of TAM with the one of its sister protein CYCA1;1 resulted in a separation-of-function allele of TAM, which prevents the partitioning of TAM to SGs while restoring a wild-type phenotype in a tam mutant background under nonheat stress conditions. Notably, plants expressing this TAM version prematurely terminate meiosis under heat resulting in unreduced gametes. Thus, the formation of TAM-containing SGs is necessary for genome stability under heat stress.

Fig. 1.. Localization of meiotic cyclins upon hsc.

Localization of (A) CYCB3;1:EGFP, (B) SDS:EGFP, and (C) TAM:mGFP in male meiocytes in leptotene (L), late leptotene-zygotene (late L-Z), zygotene-early pachytene (Z-early P), and pachytene-diakinesis (P-D) under ctcs (21°C; no heat stress) and heat shock conditions 34°C (hsc, heat stress). (D and E) Colocalization analysis of TAM:TagRFP with CDKA;1:mVenus and (F and G) with PAB4:EGFP. [(D) and (F)] Confocal images of male meiocytes during late prophase (P-D) at hsc and [(E) and (G)] corresponding pixel intensity plots across cross sections through TAM foci. (H) Localization of CDKA;1:mVenus in tam male meiocytes during late prophase (P-D) and dyad stage under ctc and at hsc. (I) Duration of CDKA;1 and TAM recruitment to SGs (in minutes). Scale bars, 10 μm. a.u., arbitrary units.

Fig. 2.. The IDR of TAM is necessary and sufficient for SG localization.

(A) Schematic representation of TAM, CYCB3;1, and mutated variants of these cyclins; IDR of TAM and CYCB3;1 and CYCA1;1 in blue/cyan/dark blue, respectively; cyclin domains of TAM and CYCB3;1 in yellow/orange, respectively; and serine (S) and alanine (A) substitution. (B) Localization of TAM:GFP in tam; delIDR:TAM:GFP in tam; and IDRswap:CYCB3;1:GFP, Aro2Ser:TAM:GFP, 15PtoA:TAM:GFP, and IDRswap:TAM:GFP in tam in male meiocytes during late prophase under ctc and hsc. Complementation analysis at ctc with confocal bright-field images of tetrad or dyads (C), Peterson-stained pollen (D), and pollen diameter quantification (in micrometers) (E). Significant differences were determined by analysis of variance (ANOVA) and Tukey post hoc test (P < 0.05). Scale bars, 10 μm [(B) and (C)] and 100 μm (D). WT, wild type.

Fig. 3.. Condensate formation of TAM.

(A and B) FRAP analysis of TAM:GFP and PAB4:eGFP in meiocytes at hsc. Representative confocal image during FRAP and normalized intensity prebleaching, bleached, and upon recovery of cytoplasmic (dotted line) and condensates (solid line) fluorescent signal. (C and D) Localization of TAM-GFP or GFP in transiently transformed Arabidopsis mesophyll protoplasts. Representative confocal images of GFP fluorescence of protoplasts expressing TAM-GFP (C) or GFP (D) under ctc and hsc and quantification of SG formation in percent; white bar, cells without SGs; gray bar, cells with at least five SGs. The absolute sample size is given in the corresponding bar. Data were collected in at least five independent transformation experiments. (E) Colocalization of TAM-GFP and PAB8-mCherry in transiently transformed Arabidopsis mesophyll protoplasts. Representative confocal images of a protoplast showing coexpression of TAM-GFP and PAB8-mCherry after incubation at hsc and fluorescence intensity plots of GFP (green)– and mCherry (magenta)–derived fluorescence. Scale bars, 10 μm [(A) to (E)] and 1 μm [recovery images, (A) and (B)].

Fig. 4.. Pollen analysis of the separation of function tam allele under hsc.

Peterson-stained anthers and pollen that come from heat-shocked flower buds containing anthers in meiotic stages (A) and quantification of pollen diameter (in micrometers) per flower (B). Significant differences were determined by ANOVA and Tukey post hoc test (P < 0.05). Scale bars, 100 μm.

Fig. 5.. Exit after meiosis I in the separation of function tam allele under hsc.

(A) Confocal images extracted from live-cell imaging of TAM:GFP TagRFP:TUA5 tam and IDRswap:TAM:GFP TagRFP:TUA5 tam male meiocytes. T, temperature (from movies S3 and S5). (B) Quantification of the ratio of premature meiotic exit (dyad formation; dark gray) and no premature exit (tetrad formation; white) of TAM:GFP tam and IDRswap:TAM:GFP tam at ctc and hsc, n = cells analyzed. MI, meiosis I.

Fig. 6.. Hypothetical model of potential mechanisms for TAM association with meiotic SGs under hsc.

Two potential mechanisms that are not mutually exclusive: TAM removal from cytoplasm (A) and TAM active control in SGs (B). At the onset of hsc, TAM (and CDKA;1 and PAB4/6) sequestration in SGs until heat stress response (hsr) is launched. TAM could have a role in translation regulation (with PAB4 and PAB6) and/or control of other substrates in SGs (no supporting experimental data provided). (C) The separation of function allele IDRswap:TAM becomes inactive in the cytoplasm under hsc and, by this, fails to phosphorylate substrates, which are required for meiosis I to meiosis II transition, and, by this, triggers meiotic exit.