Volume 241, Issue 1 p. 180-196
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Open Access

Meiotic instability and irregular chromosome pairing underpin heat-induced infertility in bread wheat carrying the Rht-B1b or Rht-D1b Green Revolution genes

András Cseh

András Cseh

HUN-REN, Centre for Agricultural Research, 2462 Martonvásár, Brunszvik u. 2, Hungary

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Andrea Lenykó-Thegze

Andrea Lenykó-Thegze

HUN-REN, Centre for Agricultural Research, 2462 Martonvásár, Brunszvik u. 2, Hungary

Doctoral School of Biology, Institute of Biology, ELTE Eötvös Loránd University, Egyetem tér 1-3, Budapest, 1053 Hungary

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Diána Makai

Diána Makai

HUN-REN, Centre for Agricultural Research, 2462 Martonvásár, Brunszvik u. 2, Hungary

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Fanni Szabados

Fanni Szabados

HUN-REN, Centre for Agricultural Research, 2462 Martonvásár, Brunszvik u. 2, Hungary

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Kamirán Áron Hamow

Kamirán Áron Hamow

HUN-REN, Centre for Agricultural Research, 2462 Martonvásár, Brunszvik u. 2, Hungary

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Zsolt Gulyás

Zsolt Gulyás

HUN-REN, Centre for Agricultural Research, 2462 Martonvásár, Brunszvik u. 2, Hungary

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Tibor Kiss

Tibor Kiss

HUN-REN, Centre for Agricultural Research, 2462 Martonvásár, Brunszvik u. 2, Hungary

Food and Wine Research Institute, Eszterházy Károly Catholic University, Eszterházy tér 1, Eger, 3300 Hungary

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Ildikó Karsai

Ildikó Karsai

HUN-REN, Centre for Agricultural Research, 2462 Martonvásár, Brunszvik u. 2, Hungary

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Blanka Moncsek

Blanka Moncsek

HUN-REN, Centre for Agricultural Research, 2462 Martonvásár, Brunszvik u. 2, Hungary

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Edit Mihók

Edit Mihók

HUN-REN, Centre for Agricultural Research, 2462 Martonvásár, Brunszvik u. 2, Hungary

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Adél Sepsi

Corresponding Author

Adél Sepsi

HUN-REN, Centre for Agricultural Research, 2462 Martonvásár, Brunszvik u. 2, Hungary

Author for correspondence:

Adél Sepsi

Email: [email protected]

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First published: 10 September 2023

Summary

  • Mutations in the Rht-B1a and Rht-D1a genes of wheat (Triticum aestivum; resulting in Rht-B1b and Rht-D1b alleles) cause gibberellin-insensitive dwarfism and are one of the most important elements of increased yield introduced during the ‘Green Revolution’.
  • We measured the effects of a short period of heat imposed during the early reproductive stage on near-isogenic lines carrying Rht-B1b or Rht-D1b alleles, with respect to the wild-type (WT).
  • The temperature shift caused a significant fertility loss within the ears of Rht-B1b and Rht-D1b wheats, greater than that observed for the WT. Defects in chromosome synapsis, reduced homologous recombination and a high frequency of chromosome mis-segregation were associated with reduced fertility. The transcription of TaGA3ox gene involved in the final stage of gibberellic acid (GA) biosynthesis was activated and ultra-performance liquid chromatography–tandem mass spectrometry identified GA1 as the dominant bioactive GA in developing ears, but levels were unaffected by the elevated temperature.
  • Rht-B1b and Rht-D1b mutants were inclined to meiotic errors under optimal temperatures and showed a higher susceptibility to heat than their tall counterparts. Identification and introduction of new dwarfing alleles into modern breeding programmes is invaluable in the development of climate-resilient wheat varieties.

Introduction

Bread wheat (Triticum aestivum) is one of our major food commodities, with a global production rate close to 1 billion tonnes per year and a world trade greater than any other crops (FAOSTAT, http://www.fao.org/faostat). The high yield performance of today's wheats greatly relies on their semidwarf stature, whereby the reduced stem elongation is associated with an improved resistance to lodging and a higher number of grains per spike (Borner et al., 1993; Miralles et al., 1998).

The semidwarf growth habit is known to be largely controlled by mutant alleles of the reduced height (Rht) genes, introduced into modern wheat varieties during the Green Revolution (1950s). The Rht-B1b and Rht-D1b alleles encode truncated repressor proteins termed as DELLAs that counteract the effect of the growth stimulant plant hormone gibberellic acid (GA) via transcriptional regulation (Hedden, 2003; Lumpkin, 2015). Wild-type (WT) DELLAs are proteolytically degraded in the presence of GA, if it binds its soluble receptor protein GIBBERELLIN-INSENSITIVE DWARF1 (GID1). Mutated DELLAs, however, are resistant to GA-GID1-dependent proteolysis and thus exert a permanent growth repression (Peng et al., 1999; Wu et al., 2011; Van De Velde et al., 2021).

The yield benefits of gibberellin-insensitive semidwarfs rely on optimal growth conditions (Rebetzke et al., 2014; Jatayev et al., 2020; Ingvordsen et al., 2022). Warm temperatures reduce wheat productivity by affecting almost all aspects of reproductive development. For instance, meiosis, the specialised cell division responsible for functional gamete formation, has a strict requirement for temperature (Bayliss & Riley, 1972; De Storme & Geelen, 2014; Bomblies et al., 2015), with the early stages being particularly heat-sensitive (Bennett et al., 1973; Draeger & Moore, 2017). At prophase I, stage of early meiosis, maternal and paternal homologous chromosomes mutually recognise each other, juxtapose by the proteinaceous structure of the synaptonemal complex (SC) and exchange parts of their genetic material via DNA crossing over (Schwarzacher, 2003; Osman et al., 2011; Mercier et al., 2015; Zickler & Kleckner, 2015). In addition to ensuring genetic diversity, meiotic recombination by crossovers (COs) is essential for creating physical connections between the homologous chromosomes, cytologically termed as chiasmata, which are central to balanced chromosome segregation in anaphase I (Hunter, 2015; Lambing et al., 2017). Recombination is initiated by a large number of programmed double-strand breaks (DSBs), which can be repaired by CO- or noncrossover (NCO) pathways (Grelon et al., 2001; Hartung et al., 2007; Drouaud et al., 2013; Benyahya et al., 2020). CO designation is regulated by multiple mechanisms with the SC playing a central role (Barakate et al., 2014; Higgins et al., 2014; Woglar & Villeneuve, 2018; Durand et al., 2022). In leptotene (the first substage of meiotic prophase) concomitant with DSB initiation, axial element (AE) proteins are loaded into the chromosome axes (Armstrong et al., 2002; Phillips et al., 2012; Hesse et al., 2019; Lambing et al., 2020). Subsequently, in zygotene AEs become interconnected by transverse filament proteins giving rise to the central element (CE) of the SC (Higgins et al., 2005; Osman et al., 2006; Khoo et al., 2012). In cereals, the SC is initially polymerised starting from the subtelomeric regions and progressing towards the interstitial regions of the chromosomes (Higgins et al., 2012), and interstitial synapsis elongates later from multiple chromosomal sites (Lenykó-Thegze et al., 2021). A meiosis-specific chromatin dynamics is involved in creating a spatially polarised chromatin organisation before SC initiation in wheat (Sepsi & Schwarzacher, 2020). Telomeres become associated at the nuclear periphery and form the telomere bouquet (Bass et al., 2000), while centromeres associate at the opposite nuclear pole and spatially restrict pericentromeric regions from the subtelomeres (Martinez-Perez et al., 2003; Sepsi et al., 2017).

Heat stress has been shown to induce meiotic instability (Draeger & Moore, 2017; Schindfessel et al., 2021; Fu et al., 2022) by delaying CO maturation (De Jaeger-Braet et al., 2022), altering CO positioning and frequency (Higgins et al., 2012; Phillips et al., 2015; Modliszewski et al., 2018) and by disrupting homologous synapsis (De Storme & Geelen, 2020; Ning et al., 2021). Meiotic irregularities result in abnormal gamete formation and infertility (Modliszewski & Copenhaver, 2015; Yang et al., 2021).

Gibberellic acid-insensitive Rht alleles are present in a large proportion of modern wheat varieties world-wide. Uncovering the vulnerability of plants carrying GA-insensitive Rht alleles is of great interest for crop breeding due to current rising temperatures, which already impose a critical threat to wheat productivity (Asseng et al., 2015; Jacott & Boden, 2020).

Elevated temperatures increase GA levels in the vegetative tissues of GA-insensitive wheats resulting in higher GA levels compared with the WT (Pinthus et al., 1989). Bioactive GAs and functional DELLA proteins are essential for reproductive development in plants (Cheng et al., 2004; Plackett et al., 2014; Jin et al., 2022) and are thus required to ensure fertility. While under optimal temperature conditions GA levels are differentially regulated in the vegetative and the reproductive tissues of the GA-insensitive DELLA mutant wheats (Webb et al., 1998), it is unclear, how early heat stress affects GA concentrations in the floral organs and how it correlates to fertility.

The present study examined the impact of a transient and moderate heat stress applied before the onset of meiosis. Meiosis I and II chromosome segregation and critical early meiotic events of Rht-B1b or Rht-D1b mutant wheats, including SC formation and recombination, were investigated in detail. Meiotic instability and infertility of Rht mutants has been discussed. To understand the effect of heat stress on GA levels in the wheat spike, we studied the transcriptional activity of the TaGA3ox gene, encoding the enzyme involved in the last stage of GA biosynthesis. Direct hormone measurements by UPLC-MS elucidated the effect of heat on the bioactive GA products in the developing wheat ear. Implications of our results for future breeding strategies and food security are discussed.

Materials and Methods

Plant material

Seeds of the ‘Maris Huntsman’ Rht-B1b-, Rht-D1b- and WT wheat (Triticum aestivum L.) near-isogenic lines (accession nos.: W9983, W9984 and W9982, respectively) were provided by the Germplasm Resources Unit of John Innes Centre (Norwich, UK). Near-isogenic lines were tested upon receipt for the Rht backgrounds by molecular markers as described by Ellis et al. (2002; Supporting Information Fig. S1). Experiments were carried out in growth cabinets at the Phytotron Facility of the Centre for Agricultural Research (ATK, Martonvásár, Hungary, 47°18′51″N, 18°46′57″E, altitude 110 m) in three successive replications in 2019, 2020 and 2021.

Temperature regimes

Following a 6-wk vernalisation period (4°C, 10 h : 14 h, light : dark), plants were potted in 12 cm × 12 cm × 18 cm pots and transferred to a growth cabinet (PGR-15; Conviron, Winnipeg, Manitoba, Canada) following the ‘T1’ spring programme (Tischner et al., 1997). Plants were transferred to a stress cabinet (PGR-15) when the main shoot entered meiotic interphase (see Methods S1). Heat stress involved a day : night temperature of 30°C for 24 h. Plants were watered twice during heat treatments.

Fertility assessment

Seed set was determined by counting the total number of grains per primary ear. Spikelet number was recorded for each ear analysed, and spikelet fertility was determined by counting the number of grains per spikelet.

Immunolabelling

Anthers were fixed in 4% PFA containing 0.5% (v/v) Igepal CA-630 (18 896; Sigma-Aldrich) for 14 min, the first 5 min involving vacuum infiltration. Meiocytes were slide-mounted and processed as described by Sepsi et al. (2018). Primary antibodies applied in the present study included a rabbit antibody raised against the N terminus of the wheat centromeric histone H3 protein (CENH3; Sepsi et al., 2017), a rat anti-ZYP1 antibody (Higgins et al., 2005), a guinea pig anti-ASY1 antibody (Desjardins et al., 2020) and a rabbit anti-γH2AX antibody (H2A.XS139p, C15310223; Diagenode, Belgium, Europe). Primary antibodies were diluted at a ratio of 1 : 300 (CENH3, ZYP1, γH2AX) and 1 : 1000 (ASY1). The list of secondary antibodies used in this study is presented in Table S1.

In situ hybridisation

The wheat centromeric retrotransposon (CRW; Li et al., 2013) and the universal plant telomeric repeat (TRS; Schwarzacher & Heslop-Harrison, 1991) were amplified by PCR and labelled by nick-translation (AF488 NT Labeling Kit; AF594 NT Labeling Kit; Jena Bioscience, Jena, Germany). In situ hybridisation followed the procedure described by Lenykó-Thegze et al. (2021) with minor modifications. The hybridisation mix contained 60% (v/v) deionised formamide (F9037; Sigma-Aldrich) and 10% (w/v) dextran sulphate (67 578; Sigma-Aldrich) in 2× SSC (saline-sodium citrate). A volume of 17 μl per slide was completed with 40–60 ng of the labelled probes. The ImmunoFISH procedure was carried out as described by Sepsi et al. (2018).

Confocal microscopy

Confocal microscopy was carried out using a TCS SP8 confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany). A series of confocal images was obtained using a HC PL APO CS2 639/1.40 oil immersion objective. Full details of confocal imaging are available in Methods S2.

Quantitative reverse transcription-polymerase chain reaction

Total RNA from the excised ears was extracted by using the Qiagen RNeasy plant mini kit after Trizol extraction, with an extra step of DNase treatment programmed in the QIAcube equipment (Qiagen Ltd). The cDNA transcription was performed with 1.0 μg of total RNA using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific Inc, Waltham, MA, USA). Reverse transcription-polymerase chain reaction (RT-qPCR) was carried out in a Rotor-Gene Q equipment (Qiagen Ltd) applying the SYBR Green technology, as described previously (Kiss et al., 2017).

The TaGA3ox2 gene has been selected to monitor TaGA3ox activities in the developing wheat ears based on its high expression levels in young reproductive tissues (Appleford et al., 2006; Pearce et al., 2011). TaGA3ox2 gene-specific primers were designed from the conserved regions of cDNA sequences derived from the three genomes of hexaploid wheat (Pearce et al., 2011; Liu et al., 2013). Reference gene Ta30797 was selected on account of its high stability in temperature treatment analyses of the floral organs (Paolacci et al., 2009). Ears of three plants were pooled for one biological replicate. Three biological replicates were collected, and two technical replicates were used in the analysis.

Droplet digital PCR

Basal and apical ear regions were separately collected to obtain three biological replicates. The ddPCR was performed by using the QX200 Droplet Digital PCR System (Bio-Rad) as described previously (Gulyás et al., 2022). Droplets were prepared by the QX200 Droplet Generator (Bio-Rad) according to the manufacturer's protocol. The water-in-oil emulsions (40 μl) were amplified on an Applied Biosystem Veriti 96 Well (Applied Biosystem, Waltham, MA, USA). Droplets were analysed by the QX200 Droplet Reader and the QuantaSoft software (Bio-Rad) according to the manufacturer's protocol.

Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS)

We developed and validated an in-house method for the most optimal SPE cartridge and conditions as described in Methods S3.

For UPLC separation, a Waters Acquity I class UPLC system (Waters, Milford, MA, USA) was used, and separation was achieved on a Waters Acquity HSS T3 column (1.8 μm, 100 mm × 2.1 mm), kept at 40°C. Mobile phase A was water containing 0.1 v/v % formic acid (FA), while mobile phase B was acetonitrile containing 0.1 v/v % FA. The flow was 0.4 ml min−1, and the gradient profile was as follows: 0 min, 5% B; from 0 to 3 min, linear gradient to 20% B; from 3 to 4.3 min, isocratic 20% B; from 4.3 to 9 min, linear gradient to 45% B; from 9 to 11 min, linear gradient to 100% B; from 11 to 13 min, kept at 100% B; from 13.01 to 15 min, back to the initial conditions of 5% B. The injection volume was 1.5 μl for all samples, which were kept at 8°C in the autosampler during the analysis.

Tandem mass spectrometric detection was performed on a Waters Xevo TQ-XS equipped with a UniSpray™ source (UniSpray Systems, Cambridge, UK) operated in timed multiple reaction monitoring (MRM) mode with the following settings: impactor voltage was 2 kV in both positive and negative modes; nebuliser gas, 6 bar; desolvation temperature, 550°C; cone gas flow, 450 l h−1; desolvation gas flow, 1000 l h−1. For collision gas, argon (5.0 purity) was used with a gas flow of 0.15 ml min−1. Unit resolution was applied to each quadrupole. Dwell time set to be automatically calculated to take at least 20 points across each peak for quantitation. Where possible, at least three MRM transitions were used for data acquisition and the transition having the highest S : N ratio was used for quantitation (Table S2). Data processing was done using Waters MassLynx 4.2 and TargetLynx software. Besides the internal standard, QC samples were also used to verify method performance in each batch to monitor the recovery rates of compounds.

Statistical analyses

Statistical analyses were carried out using the open-source statistical analysis program JASP 0.16 (University of Amsterdam, Amsterdam, the Netherlands). Normality of data was tested by Saphiro–Wilk test, while equality of variances was verified by Levene's test. Figures were produced using the R statistical platform (v.3.6.3) via R studio software (2022.02.0 + 443 ‘Prairie Trillium’), and the ggplot2 package (3.4.1) was used for visualisation. Further details on the statistical tests are described in Methods S4.

Results

A short period of elevated temperature reduces fertility of Rht-B1b and Rht-D1b mutants

Elevated temperature resulted in a significantly lower mean seed set per primary spikes for both Rht-B1b and Rht-D1b lines compared with a nonsignificant decrease in WT (ANOVA, F(5, 172) = 2377.9, ω2 = 0.42, P < 0.001; Figs 1a,b, S2). Spikelet number did not vary with genetic background, or the temperature treatments applied in this study (Fig. S3), indicating that reduced spikelet fertility was responsible for the seed set reduction (Fig. 1c; Kruskal–Wallis, Dunn's post hoc test, H(5) = 76.829, P < 0.001). The main spikes of Rht-B1b and Rht-D1b mutants exhibited a 20% and 31% loss in spikelet fertility. A pronounced effect (40% loss) was detected within the basal region of Rht-D1b ears (Fig. 1d,e; Kruskal–Wallis, Dunn's post hoc test, H(5) = 66.899, P < 0.001), whereas the apical region exhibited a 22% reduction (Kruskal–Wallis, Dunn's post hoc test, H(5) = 50.292, P < 0.001). Rht-B1b in turn showed a significant but uniform loss of c. 20% within both regions (Fig. 1d,e; P < 0.001 in both cases). The early reproductive stage of Rht-B1b and Rht-D1b lines thus exhibits a higher vulnerability to heat stress compared with the WT (tall) line, with the basal region of Rht-D1b ears being particularly heat-sensitive.

Details are in the caption following the image
Effect of heat stress on the fertility of Rht-B1b and Rht-D1b mutant wheats (semidwarfs) with respect to the wild-type (WT; tall). (a) Schematic representation of the heat stress treatment relative to the developmental stage of bread wheat. Approximate timing of wheat development under Central European field conditions is indicated under the respective developmental stages. Heat stress (30°C) was applied during early reproductive development encompassing the interphase immediately preceding meiotic prophase. At anthesis, natural cross-pollination was prevented by using pollinator bags. Fertility was assessed after grain harvest at full maturity. Artwork has been prepared in Canvas Draw. (b) Seed set is reduced by heat stress in the primary ears of the Rht-B1b and Rht-D1b semidwarf mutants. Counts of total seeds per primary ears are shown (data points shown on the right side) for each genotype and treatments (21°C and 30°C) together with the corresponding plant heights (cm, data points on the left side). Data points connected by a line originate from the same plant (Statistical analysis shown in Supporting Information Fig. S2). (c) A short period of heat stress (30°C) reduces spikelet fertility (seed per spikelet) within the primary ears of Rht-B1b and Rht-D1b semidwarf near-isogenic lines (‘Maris Huntsman’ winter wheat series, see panel (e). Boxplots show counts of seed per spikelet with mean values (horizontal lines) ± SD (whiskers). The number of plants sampled for each line and treatment is shown in brackets. ***, P < 0.001 (Kruskal–Wallis test followed by Dunn's post hoc test). (d) Heat-treated Rht-B1b and Rht-D1b wheat lines show similar reduction in spikelet fertility within the apical region of the ears. Counts of seed per spikelet within the apical region of primary ears are presented as mean values ± SD for all three genotypes grown under optimal (20°C) and elevated temperature (30°C) conditions. ***, P < 0.001 (Kruskal–Wallis test followed by Dunn's post hoc test). (e) Both Rht-B1b and Rht-D1b show a significant reduction in fertility in the basal region of the primary ears after heat stress, in contrast to the WT. Heat-treated Rht-D1b shows a dramatic reduction in fertility within the basal region. Counts of seed per spikelet are presented as mean values ± SD. ***, P < 0.001 (Kruskal–Wallis test followed by Dunn's post hoc test).

High temperature increases the frequency of chromosome mis-segregation in Rht mutants

To discover whether heat-induced fertility losses may arise from errors in the meiotic process, we investigated the effect of heat stress on anaphase I and anaphase II chromosome segregation and on genome stability in tetrads and microspores. We combined DAPI counterstaining with CENH3 immunolabelling, which marks functional centromeres (Henikoff, 2001; Houben & Schubert, 2003) and followed chromosome behaviour together with centromere activity.

Wild-type PMCs grown under optimal temperatures revealed balanced homologous chromosome segregation at anaphase I (Fig. 2a), followed by the segregation of the chromatids at anaphase II and meiosis II, which resulted in four sets of chromatids arranged in an isobilateral tetrad configuration (Fig. 2a). Chromosome mis-segregation and chromatin elimination by micronuclei were detected from anaphase I to the early microspore stage in the heat-treated WT, Rht-B1b and Rht-D1b mutant lines. Unexpectedly, equivalent meiotic errors were detected in the control Rht-B1b and Rht-D1b meiocytes, although with lower frequencies than in the heat-treated cells. CENH3 immunofluorescence revealed mis-segregating chromosomes equipped with functional centromeres, although random positioning around the bulk chromatin was frequent (Fig. 2a). Analysis of contingency tables followed by chi-squared tests showed unequal incidence of aberrant cells within the investigated WT and Rht mutant genotypes and between their respective temperature treatments (χ2(5) = 148.654, P < 0.001). Binomial test revealed that WT male meiocytes exhibited a significantly higher (15%, P < 0.001) chromosome mis-segregation under heat stress than under optimal temperature. A similar frequency of aberrant anaphase-telophase I and MII cells occurred within the Rht-B1b and Rht-D1b controls than the wild treated (P = 0.373 and 0.160 vs WT treated, respectively; Fig. 2b; Table S3). Rht-B1b and Rht-D1b mutants subject to heat stress showed a further increase in the frequency of mis-segregating chromosomes (20%, P < 0.001 and 22%, P < 0.001, respectively). Cytological analysis on the WT control showed that microspores progressed to the mid-uninucleate stage and produced normal microspores (an eccentric nucleus enclosed by the microspore wall; Fig. 2c), while a higher number of aberrant microspores occurred within the heat-stressed Rht-B1b and Rht-D1b lines compared with their controls (χ2(5) = 37.416, P < 0.001). Aberrations were manifested as deformed nuclei or as multiple micronuclei located around the main nucleus (Fig. 2c; Table S4). Heat-inferred fertility losses may thus be the result of unbalanced chromosome segregation and programmed chromatin elimination throughout MII where the frequency of aberrations was significantly higher in the mutants than in the WT.

Details are in the caption following the image
Heat stress induces a considerable chromosome mis-segregation in Rht-B1b, Rht-D1b mutant wheat leading to aberrant meiotic products. (a) Meiotic progression of Rht-B1b, Rht-D1b and wild-type (WT) cells arising from heat-stressed (30°C) or optimal (21°C) temperature conditions. Meiotic stages from top to bottom. Anaphase I, Interphase II, Anaphase II and the tetrad stage. Chromatin is counterstained with 4′,6-diamidino-2-phenylindole (DAPI, grey), and centromere-specific histone H3 (CENH3, red) protein immunofluorescence indicates the location of functional centromeres. Arrows indicate typical examples of lagging chromosomes and chromatin elimination. An amorphous nucleus is circled in yellow. Bars, 5 μm. (b) Frequency of meiotic errors scored from heat-treated and control Rht-b1b, Rht-D1b and WT meiocytes encompassing stages from anaphase I to tetrads. The number of cells sampled for each line and treatment is shown in brackets. (c) Examples of chromatin fragmentation observed in uninucleate wheat microspores originating from heat stress. Chromatin is shown by DAPI counterstaining (white), and the microspore wall (blue) is visualised by capturing autofluorescence by laser scanning microscopy. Micronuclei are marked with arrows, and an amorph nucleus is encircled in yellow. A representative uninucleate microspore sampled from a WT control plant is presented for comparison. The frequency of aberrant microspores is indicated for each genotype and treatment. The number of cells sampled for each line and treatment is shown in brackets. Bars, 5 μm.

Rht mutants show a marked reduction in crossover frequency after heat stress

Heat stress is known to affect CO frequency and distribution (Bomblies et al., 2015; Modliszewski & Copenhaver, 2015). We therefore aimed to determine heat-induced changes in CO frequency in Rht-B1b and Rht-D1b mutant plants by scoring the number of chiasmata per meiotic metaphase I (MI) spread (Sybenga, 1975). In situ hybridisation (Schwarzacher et al., 1989), utilising wheat centromeric retrotransposons (CRW; Li et al., 2013) and the TRS (Schwarzacher & Heslop-Harrison, 1991) as probes visualised centromere and telomere positions to show bivalent orientation. Under optimal conditions, WT MI spreads exhibited 51 chiasmata on average (SD = 4.4, n = 22), the large majority (92%) of which were ring bivalents (Fig. 3a; Table S5). This did not vary from chiasma frequencies measured in the Rht-B1b and Rht-D1b control MI cells (Rht-B1b M = 53, SD = 3.8, n = 25; Rht-D1b M = 53, SD = 4.0, n = 21; Kruskal–Wallis, Dunn's post hoc test, H(5) = 78.173, P < 0.001). Heat stress however significantly reduced chiasma number in all three genotypes causing a 12% reduction in the WT (M = 45, SD = 8.2, n = 17; P = 0.01), a loss of 38% in Rht-B1b (M = 33, SD = 6.2, n = 21; P < 0.001) and a reduction of 21% in Rht-D1b (M = 42, SD = 4.6, n = 18; P < 0.001; Fig. 3a). This was consistent with a significant increase in the number of rod bivalents (Fig. 3a; Table S5; Kruskal–Wallis, Dunn's post hoc test, H(5) = 59.602, P < 0.001). Crossover assurance (Desjardins et al., 2020, 2022; Higgins et al., 2022; Pochon et al., 2022) was impaired, seen in the appearance of univalent chromosomes (Figs 3, S4; Table S5). Contingency tables followed by Chi-squared tests indicated a nonequal incidence of cells with univalent chromosomes within the control and heat-treated meiocytes of the WT, Rht-B1b and Rht-D1b lines (χ2(5) = 26.452, P < 0.001; Fig. 3a). Binomial tests revealed that heat stress significantly increased the frequency of cells carrying univalent chromosomes in all three genotypes (Fig. 3a,b; P < 0.001 in all three cases). In addition, meiocytes from Rht-B1b and Rht-D1b treated plants exhibited a higher frequency of univalents compared with WT (P < 0.001; P = 0.02, respectively). Reduction in the homologous recombination frequency was accompanied by the occurrence of nonlegitimate connections between the bivalents (Figs 3a, S4S5). This was clear from poorly spread chromosome conformations connected by thin chromatin bridges, often hindering regular chromosome alignment and centromere biorientation at the equatorial plate (Figs 3a, S4, S5).

Details are in the caption following the image
Effect of heat stress on chiasma frequency and number of double-strand breaks (DSBs) in the Rht-B1b and Rht-D1b mutant wheats. (a) Fluorescence in situ hybridisation on meiotic metaphase chromosomes of control and heat-treated Rht-B1b, Rht-D1b and wild-type (WT) plants reveals the location of the centromeres (the wheat centromeric retrotransposon CRW, red) and telomeres (the universal plant telomeric repeat sequence TRS, blue) and together with DAPI counterstaining (grey) identifies chromosome orientation. Bars, 5 μm. Heat stress dramatically reduces the number of chiasmata per cell (presented by an interval plot; dots denote mean values, error bars show ± SD; *, P = 0.05; ***, P < 0.001) by promoting rod bivalent formation at the expense of ring bivalents (an interval plot shows mean values ± SD; ***, P < 0.001) and leads to the loss of obligate chiasmata (univalents) in both Rht-B1b and Rht-D1b plants. The number of cells carrying univalent chromosomes across the genotypes and treatments used in the study is presented in a bar plot. (b) Co-immunofluorescence labelling of ASY1 protein and γ H2AX foci on Rht-B1b, Rht-D1b and WT wheat meiocytes collected from optimal and heat-treated conditions. Left panel: The number of DSBs measured in the WT, Rht-B1b and Rht-D1b meiocytes are presented as mean values (dots) ± SD (error bars). **, P = 0.01 (Kruskal–Wallis test followed by Dunn's post hoc test). The number of DSBs is reduced following heat stress in the WT but remains unaffected in the Rht-B1b and Rht-D1b mutants. Right panel: Microscopic images show ASY1 and γH2AX immunosignals in monochrome (first two columns) and pseudocoloured on Merge (red and white, respectively). γH2AX foci are surface rendered on Merge. Bars, 5 μm.

Double-strand breaks are unaffected by heat in Rht-B1b and Rht-D1b mutant wheats

We next investigated whether the heat-induced reduction in chiasma frequency correlated with a lower number of DSBs. DSB quantification was conducted on leptotene nuclei by co-immunofluorescence using anti-TaASY1-antibody as a reliable marker for meiotic timing, and anti-γH2AX antibody marking DSB sites (Fig. 3b; Lang et al., 2012). Heat stress reduced the number of γH2AX loci by 19% in the WT (control M = 1038, SD = 274, n = 23; heat-treated M = 844, SD = 231, n = 23; Kruskal–Wallis, H(5) = 20.567, Dunn's post hoc test, P = 0.005). Rht-B1b and Rht-D1b mutants exhibited a lower number of γH2AX loci (26% and 27% lower, respectively) under optimal temperature compared with the WT control (P < 0.001 in both cases). No further decrease was however detected after heat treatment in any of the Rht mutants (P = 0.207 and 0.482; Fig. 3b). This suggested that normal DELLA proteins are required for the modulation of DSB numbers upon high temperature stress in wheat.

Axial element loading after heat stress in the WT and the Rht-B1b and Rht-D1b mutants

We sought to investigate whether the reduction in CO number and erroneous chromosome conformations observed at metaphase were associated with errors in chromosome axes. We therefore used immunofluorescence to visualise TaASY1, a protein associated with the meiotic chromosome axes, essential for SC formation and CO assurance (Armstrong et al., 2002; Lambing et al., 2020; Pochon et al., 2022). Pollen mother cells (PMCs) of the WT originating from optimal and elevated temperatures revealed normal AE linearisation at leptotene, gradual ASY1 depletion at zygotene and disappearance of the ASY1 signal from the synapsed axes by pachytene (Fig. S6; Sepsi et al., 2017; Desjardins et al., 2020; Osman et al., 2021). ASY1 loading appeared normal in the mutant lines at 21°C and after heat treatment (Figs 4, 5), but ASY1 could still be observed on a subset of heat-treated Rht-B1b and Rht-D1b pachytene stage meiocytes colocalising with the synapsed axes (Figs 4b, 5b). Heat stress thus did not affect axis formation in the WT, but the presence of ASY1 on the synapsed axes in pachytene suggested delayed or incomplete protein depletion.

Details are in the caption following the image
Axial element (AE) formation and progression of synapsis in meiotic prophase I of control and heat-stressed Rht-B1b mutant wheat plants. (a) Co-immunofluorescence of synaptonemal complex (SC) AE protein ASY1 (red) and transverse filament protein ZYP1 (blue) shows linear AEs on the control leptotene meiocytes and marked SC polymerisation from the chromosome ends, forming a conical structure at the nuclear periphery in early-zygotene (Early-Zyg). Proximal chromosomal regions contain multiple ZYP1 foci. ZYP1 lengthening along the chromosome arms occurs from mid-zygotene (Mid-Zyg) to late zygotene (Late Zyg), and synapsis is complete by pachytene. ZYP1 signal is uneven with some regions showing discontinuous threads (Mid-late-zygotene, 21°C). (b) Synapsis initiation is altered in the stressed meiocytes, with very few ZYP1 foci outside the subtelomeric regions (see early-zygotene at 30°C). Noncontinuous ZYP1 axes show abnormal SC structure after heat stress from mid-zygotene to pachytene. Bars, 5 μm.
Details are in the caption following the image
Axial element (AE) formation and progression of synapsis in meiotic prophase I of Rht-D1b mutant control and heat-stressed wheat plants. (a) Co-immunolocalisation of synaptonemal complex (SC) AE protein ASY1 (red) and transverse filament protein ZYP1 (blue). A representative leptotene nucleus shows linear ASY1 signal denoting AEs formed along the chromosomes. In early-zygotene (Early-Zyg), short ZYP1 stretches are dispersed at the periphery of the nucleus indicating SC elongation. Multiple punctate ZYP1 foci are dispersed and indicate additional initiation points. ZYP1 elongates between the chromosome arms from mid-zygotene (Mid-Zyg) to late zygotene (Late Zyg), and synapsis is complete by pachytene. ZYP1 signal is irregular with discontinuous threads (Mid-late-zygotene at 21°C). (b) Synapsis initiation is altered in the stressed Rht-D1b meiocytes, with a loss of the characteristic early spatial asymmetry (see dispersed signal on early-zygotene at 30°C). Discontinuous ZYP1 axes show abnormal SC structure after heat stress from mid-zygotene to pachytene. Bars, 5 μm.

Heat stress perturbs synaptonemal complex formation in Rht-B1b and Rht-D1b

We next investigated the effects of heat stress on SC polymerisation in the Rht-B1b and Rht-D1b lines and compared it with WT. Immunofluorescence of ZYP1, the SC transverse filaments protein revealed the nuclear localisation of the SC central element and indicated synapsed chromosomal regions (Higgins et al., 2005). Normal SC morphogenesis in the control WT and Rht-B1b meiocytes was evident from subtelomeric SC initiation (Sepsi et al., 2017; Desjardins et al., 2020; Osman et al., 2021) and numerous punctate signals or short ZYP1 segments within the interstitial regions (Figs S6, 4a). These become elongated during mid-zygotene, and synapsis was perfect by pachytene (Figs S6, 4a). Unexpectedly, Rht-D1b meiocytes lacked the spatial asymmetry characteristic of early meiosis in cereals (Higgins et al., 2012; Sepsi et al., 2017). Instead, multiple SC initiation points were uniformly distributed in the nucleus, without any visible spatial imbalance, indicating a perturbed meiosis already under control conditions (Fig. 5a).

Heat stress further altered SC formation in both Rht-B1b and Rht-D1b mutants. In 65% of the heat-stressed Rht-B1b early-zygotene nuclei (number of sampled PMCs are summarised in Table S6), subtelomeric synapsis initiated along with enlarged ZYP1 polycomplexes, while the number of interstitial punctate SC signal was reduced (Fig. 4b). A subset of the sampled Rht-D1b early-zygotene nuclei (20%) exhibited equivalent SC initiation defects to Rht-B1b, whereas the majority (c. 50%) showed a dispersed SC initiation, reminiscent of the Rht-D1b controls (Fig. 5a,b). High-resolution microscopy revealed discontinuous SC structures in the heat-treated Rht-B1b and Rht-D1b PMCs, and to a lesser extent (c. 18% of the nuclei) in the WT. Discontinuous SC was evident from mid-zygotene to pachytene, with occasional enlarged ZYP1 polycomplexes (Figs 4b, 5b, S7). Synaptonemal complex (SC) formation thus shows an increased heat-susceptibility in the Rht-B1b and Rht-D1b lines compared with the WT, most likely due to a genetic predisposition seen by meiotic alterations under optimal conditions.

Rht mutations alter meiotic chromatin dynamics

Synaptonemal complex (SC) emergence in wheat is preceded by a polarised chromatin arrangement, where chromosomes are arranged by their telomeres and centromeres restricted to the two extremes of the nucleus. Telomere polarisation is realised by the formation of the telomere bouquet (Bass et al., 1997; Golubovskaya et al., 2011; Richards et al., 2012), which is coincident with the association of the 42 wheat centromeres into 7–14 groups in the opposite nuclear pole (Martinez-Perez et al., 2003; Sepsi et al., 2017). To reveal whether heat stress affects these chromosome dynamics, we visualised telomere and centromere behaviour during early meiosis by immunofluorescence in situ hybridisation (ImmunoFISH) followed by optical sectioning with confocal laser scanning microscopy and 3D rendering. Co-immunofluorescence of the axis protein ASY1 and the centromere-specific histone H3 protein (CENH3) followed by FISH with TRS showed telomere gathering at the nuclear periphery on late leptotene nuclei of WT plants at both 21°C and 30°C, indicative of normal telomere bouquet formation (Fig. 6). In the meiocytes of the Rht-B1b and Rht-D1b mutants, a loose telomere gathering appeared at both 21°C and 30°C, with occasional multiple minor telomere associations (Fig. 6). We measured separately nuclear volumes and the volume occupied by the telomeres for each genotype and treatment. Telomeres occupied 5.9% (n = 23) and 8.9% (n = 20) of the nuclear volumes within the WT control and heat-treated plants, respectively. In Rht-B1b control and heat-treated nuclei, telomeres localised within 16.5% (n = 14) and 16.3% (n = 28) of the nuclear volume. Similarly, telomeres in Rht-D1b control and heat-treated plants spread to 15.9% (n = 21) and 33.2% (n = 20) of the nucleus (Fig. 6), indicating a loose telomere bouquet formation compared with the WT. Regular centromere associations were uncovered at the nuclear periphery in all genotypes and treatments (Fig. 6), which dissolved into the nuclear space by early-mid-zygotene (Fig. S8), as expected (Sepsi et al., 2017). A loose telomere bouquet in the Rht mutants is thus coupled with normal centromere dynamics in the mutants, which appears to be insufficient for normal synapsis initiation after heat stress treatment.

Details are in the caption following the image
Deficient telomere bouquet formation in early prophase I of Rht-B1b and Rht-D1b mutant wheat is coupled with normal centromere dynamics. ASY1 protein labelling (red on merge) allowed the identification of leptotene meiocytes. Simultaneous in situ hybridisation with the universal telomeric repeat probe (TRS, white and surface rendered on merged) reveals normal telomere bouquet formation in the control and heat-treated WT pollen mother cells. The control and heat-treated Rht-B1b and Rht-D1b nuclei displayed a loose bouquet. On the right side of the microscopic images, the percentage of the nuclear volume occupied by the telomeres (red line) is shown with respect to the total nuclear volume (grey line). CENH3 co-immunofluorescence (blue and surface rendered on merge) denotes normal centromere associations at the nuclear periphery in each line and treatment. Bars, 5 μm.

A short period of heat stress activates the transcription of GA biosynthesis gene TaGA3ox but GA levels remain unaffected

Impaired DELLA protein degradation in GA-insensitive mutants of wheat leads to a marked increase in endogenous GA1 in the vegetative tissues but not in the developing ears (Appleford & Lenton, 1991; Webb et al., 1998). Moreover, elevated temperatures increase bioactive GA levels several fold in the vegetative tissues of GA-insensitive DELLA mutants relative to the WT. Since GA plays a critical role in the meiotic process, we sought to elucidate whether the marked negative effect of heat on the meiotic development of GA-insensitive DELLA mutants may be dependent on GA levels. We therefore measured the effect of a short period of elevated temperature on the final stage of GA biosynthesis and GA levels in the ears of tall and GA-insensitive wheats. We first investigated the transcription of TaGA 3-oxidase (TaGA3ox) gene encoding the enzyme that catalyses the final reaction of GA biosynthesis (Fig. 7a; Pearce et al., 2015; Barker et al., 2021). RT-qPCR showed significantly increased TaGA3ox transcript levels in the ears of each genotype after elevated temperature treatments (Kruskal–Wallis, H(5) = 30.5, P < 0.001; Dunn's post hoc test, WT P = 0.022, Rht-B1b P ≤ 0.001 and Rht-D1b P = 0.005; Fig. 7b). mRNA accumulation was higher within the Rht-B1b and Rht-D1b mutants (4.7- and 2.3-fold increase, respectively) compared with the WT (2.15-fold increase). Measurement of TaGA3ox transcript concentrations between the basal and apical regions of the ears by ddPCR showed a steady transcription along the spike in each genotype under control conditions (Kruskal–Wallis test, H(11) = 28.2, P = 0.003, Dunn's post hoc test: WT P = 0.166, Rht-B1b P = 0.454, Rht-D1b P = 0.176; Fig. S9). Heat stress significantly increased transcript concentrations in the basal ear region of the WT (P = 0.017; Fig. S9) and in both ear regions of Rht-B1b (P = 0.031 and 0.011, respectively) and Rht-D1b (P = 0.005 and 0.022, respectively; Fig. S9).

Details are in the caption following the image
Gibberellin biosynthesis pathway is activated by heat stress, but endogenous gibberellic acid (GA) levels remain stable following heat treatment. (a) The late stages of the gibberellin biosynthesis pathway (Hedden, 2020). (b) Relative transcript levels reveal a strong activation of the TaGA3ox gene after heat stress in the Rht-B1b and Rht-D1b mutant wheat ears. Measurements are shown as means ± SD (error bars) from three biological replicates. *, P = 0.05; **, P = 0.01; ***, P < 0.001 (Kruskal–Wallis test followed by Dunn's post hoc test). (c) Endogenous bioactive gibberellin GA1, GA3 and GA4 concentrations along with concentrations of the GA1 precursor GA20 and the GA1 metabolite GA8 in the young developing ears of control and heat-stressed wild-type, Rht-B1b and Rht-D1b wheat lines. GA1 is the predominant bioactive gibberellin present in the young developing wheat ears. The levels of GA1 concentrations did not vary significantly between the different Rht genetic backgrounds and between the different temperature treatments applied in the present study. The concentration of GA8 did not vary significantly after heat treatment. GA3, GA4 and GA20 were present at very low quantities in the young developing wheat ears, irrespective of the genotype and treatment.

Endogenous GA levels were measured by UPLC-MS–MS and the bioactive GA1, GA3 and GA4, and the inactive forms GA20 and GA8 were quantified using deuterated GAs as internal standards. GA3 and GA4 levels remained below 0.5 ng g−l fresh weight (FW) in each genotype and treatment (Fig. 7c; Table S7), indicating that they are not predominant. A similar profile was detected in the GA1 precursor GA20 (< 0.5 ng g−l FW; Fig. 7c; Table S7). Bioactive GA1 however ranged from 2 to 3 ng g−l FW without showing any significant difference between the genotypes and treatments (Kruskal–Wallis, H(5) = 7.53, P = 0.184, WT M = 2.1 ng g−l FW, Rht-B1b M = 3.1 ng g−l FW, Rht-D1b M = 2.2 ng g−l FW). The GA1 metabolite, GA8 was present in the ears of all genotypes and showed a minor, nonsignificant decline after heat treatment (Kruskal–Wallis, H(5) = 15.3, P = 0.009, Dunn's post hoc test, WT control vs WT treated P = 0.06; Rht-B1b control vs Rht-B1b treated P = 0.051, Rht-D1b control vs Rht-D1b treated P = 0.2). These indicate that in the wheat tissues measured by our analysis heat stress activated the final step of GA biosynthesis but neither the genetic background nor the heat treatment affected GA levels.

Discussion

We imposed a short transient heat stress in a series of controlled-environment experiments and showed that heat applied in the early reproductive stage has a greater effect on the fertility of semidwarf Rht-B1b and Rht-D1b wheats than on the WT. Our interest resulted from recent warming air temperatures becoming frequent in spring (https://climate.copernicus.eu/dry-and-warm-spring-and-summer), which overlaps the wheat's reproductive cycle. The substantial heat sensitivity of the Rht-B1b and Rht-D1b mutant wheats shown in the present work was consistent with earlier reports (Borner et al., 1993; Law, 1998); however, the magnitude of negative effects and the causes of their significant heat-induced infertility remained unexplored to date.

Heat reduces crossover frequency but not DSB number in Rht-B1b and Rht-D1b

In wheat, CO frequency drops significantly between 26°C and 28°C (Coulton et al., 2020) whereas Arabidopsis exhibits reduced CO rates between 28°C and 30°C (Lloyd et al., 2018; Modliszewski et al., 2018; De Storme & Geelen, 2020). In the present study, a transient 30°C heat treatment significantly decreased chiasma frequency in all the wheat genotypes, although Rht-B1b and Rht-D1b mutants exhibited a considerably greater reduction. Substantial decrease in CO rates was however not associated with a reduction in the number of recombination initiation sites in Rht-B1b and Rht-D1b, while the number of DSBs appeared to diminish in WT. This suggests an early DELLA-dependent effect in the WT, where recombination may be modulated at DSB initiation. In the mutants, however, that showed a lower number of DSBs under optimal temperatures compared with the WT, recombination is affected at a later stage, possibly during CO designation. While DELLA proteins are critical for reproductive development (Plackett et al., 2014), the molecular pathway through which gibberellin-insensitive DELLA proteins may reduce initial DSB numbers is still unclear. Increasing evidence points to GA-mediated DELLA degradation as a mechanism regulating reproductive development in plants (Cheng et al., 2004; Gomez et al., 2020). Interestingly, fluctuations in DSB numbers are not always reflected in CO frequency (Martini et al., 2006; Cole et al., 2012; Yokoo et al., 2012; Varas et al., 2015) and stable DSB numbers can be contrasted by an increase in Class I COs after a moderate heat treatment (Modliszewski et al., 2018). Final CO rates thus have a limited dependency on DSB numbers but rather become determined during DNA repair, which processes DSBs into COs at the expense or in favour of noncrossovers. DNA repair is increasingly affected by heat stress in Arabidopsis, which is reflected in perturbed homologue pairing and SC formation, a recombination-dependent prolongation of the CO maturation process with impaired resolution of recombination intermediates (De Jaeger-Braet et al., 2022; Zhao et al., 2023). The occurrence of heat-induced nonlegitimate meiotic chromosome connections, as detected in the present work, provides further basis for an altered DSB repair. Chromosome bridges are likely to represent regions in which nonhomologous recombination was initiated but repair prevented or delayed. Heat-inferred ectopic interlinks, indicating promiscuous chromosome interactions, are dependent on SPO11 in Arabidopsis (De Storme & Geelen, 2020), the DNA topoisomerase that catalyses DSB formation at the onset of meiotic prophase I (Keeney et al., 1997; Grelon et al., 2001; Da Ines et al., 2020). Heat stress thus perturbs the stringency or the accuracy of homology recognition machinery. Nonhomologous connections are allowed while repair mechanisms appear to fail resolving these connections, leading to a deficient metaphase plate and unbalanced chromosome segregation.

The equatorial position of bivalents and subsequent segregations can be affected by the loss of obligate chiasma (Desjardins et al., 2020), but additional heat-induced negative effects on the microtubule array cannot be overruled. Programmed chromosome elimination was not initiated by centromere inactivity, as micronuclei carried chromatin with still active centromeres. The number of heat-induced meiotic aberrations from anaphase I to the tetrad stage and further to microspore stage indicated that Rht-B1b and Rht-D1b mutant lines were increasingly affected by meiotic errors than the WT plants.

Heat-induced synaptic events and perturbed chromatin dynamics in Rht-B1b and Rht-D1b mutants

We explored for the first time in wheat the effects of a moderate heat stress on the localisation and structure of the SC axial and central elements, detected by ASY1 and ZYP1 immunofluorescence. In A. thaliana, SC CE assembly is altered at 30–32°C (De Storme & Geelen, 2020; De Jaeger-Braet et al., 2022) whereas AEs become only destabilised at temperatures over the fertile threshold (36–38°C, 24 h; Ning et al., 2021). These are consistent with our findings on WT wheat, where normal chromosome axis was associated with SC disruptions.

Additionally, a disorganised telomere bouquet was linked with an altered synapsis initiation in both heat-treated Rht mutant lines, including polycomplex formation and a loss of characteristic spatial asymmetry (Higgins et al., 2012; Sepsi et al., 2017; Osman et al., 2021) in Rht-D1b. Polycomplexes represent abnormal SC aggregates and are one of the most frequent heat-induced alterations observed in the SC structure (Loidl, 1989; Higgins et al., 2012; Morgan et al., 2017; Ning et al., 2021; Fu et al., 2022) and have been proposed to result from temperature-inferred denaturation of the SC proteins or prior abnormalities in the chromosome axis (Rog et al., 2017).

The heat-induced dispersed synapsis initiation in the Rht-D1b mutants, indicative of disrupted spatial asymmetry, was reminiscent of that observed in barley at temperatures in the range of 30–36°C (Higgins et al., 2012). Unexpectedly, dispersed synapsis initiation and perturbed telomere bouquet appeared in control mutant plants as well. The accumulation of meiotic abnormalities in the heat-treated Rht-B1b and Rht-D1b plants and the appearance of some of these alterations (although at lower frequencies) under optimal temperature conditions suggested a genetic predisposition for synaptic defects in the Rht mutants. A tolerable level of meiotic aberrations occurring under optimal conditions appears to be aggravated by heat stress, rising to a threshold that would have an effect on plant fertility.

Bioactive gibberellin levels are unaffected by the Rht mutations and heat stress in the developing wheat ears

In the present study, the activated transcription of TaGA3ox gene involved in the final stage of GA biosynthesis was accompanied by stable GA levels. This is in contrast with data collected from the vegetative tissues of GA-insensitive DELLA mutants where GA1 quantities show several fold accumulation vs WT (Fujioka et al., 1988; Talon et al., 1990; Appleford & Lenton, 1991; Webb et al., 1998). Bioactive GA levels thus appear to be differentially regulated between the vegetative and reproductive tissues in cereals (Fig. S10). Although in very low quantities, GA is required for normal meiotic development and fertility (Wilson et al., 1992; Goto & Pharis, 1999; Pearce et al., 2013). High-temperature-induced disrupted early reproductive development has been associated with a reduced (e.g. rice, Oryza sativa; Tang et al., 2008; Wu et al., 2016) or an increased GA concentration (e.g. maize, Zea mays; Wang et al., 2020) in the reproductive tissues. Stable GA levels however exclude GA fluctuations as a major mediator of heat stress responses in the wheat spike, at least under the short period of high temperature applied in the present study.

We show that the meiotic process, including prophase I events, is markedly affected by heat in wheat carrying truncated DELLAs. DELLA degradation is also required for floral development due to its ability to bind and release transcription factors, thus regulating transcription of target genes (Fu et al., 2002; Cheng et al., 2004; Fukazawa et al., 2021; Jin et al., 2022). Whole-genome microarray analysis following activation of the DELLA paralogue REPRESSOR OF ga1-3 (RGA) in Arabidopsis revealed c. 800 downregulated and upregulated genes during floral organ development with a considerable number being involved in phytohormone signalling or stress responses (Hou et al., 2008).

A direct effect of DELLA on meiotic process would require local DELLA expression. This is contradicted by evidence from Arabidopsis male sporogenesis where the RGA DELLA paralogue is exclusively expressed in the somatic cells surrounding the meiocytes (Liu et al., 2017). A different pattern of DELLA expression appears to emerge however within the members of the Triticeae. For instance, the rice DELLA protein SLENDER RICE 1 (SLR1) is expressed in the tapetum and the meiocytes (Hirano et al., 2008; Tang et al., 2010), and transcriptome data from cytologically staged barley meiocytes confirm DELLA expression (Barakate et al., 2021). DELLA protein-encoding genes are also expressed in wheat anthers (Pearce et al., 2011; Van De Velde et al., 2021), although it is to be determined if transcription is present in the meiocytes. These data support that transcriptional regulation by DELLA may affect the expression of meiotic regulators involved in early prophase events. Given the large number of DELLA targets, DELLA protein expression and degradation allows a rapid response to environmental stimuli by introducing an immediate cell cycle arrest or acceleration, contributing to survival under stress conditions (Cheng et al., 2004; Harberd et al., 2009; Gomez et al., 2020). GA-insensitive truncated DELLA proteins (Rht mutants) may affect heat response via their possible accumulation in the floral organs. Permanent repression or activation by DELLA accumulation may reduce the adaptability of important developmental processes. Further studies are needed to identify possible key players in DELLA downstream signalling at meiosis, and its interplay with hormonal networks involved in stress responses and fertility in cereals.

During the present work, the extent of heat stress was specifically selected to model the effects of a single heat wave on the meiotic cell division and fertility of wheat, by focussing on the Green Revolution semidwarf genetic backgrounds, which occur in most of today's wheat cultivars. Our study suggests that under the warming climate, vulnerability of current crop varieties needs to be revisited and addressed globally. Among these, thermotolerance during the period of floral development needs a primary focus, due to the temperature sensitivity of the production of viable gametes. Further research on novel dwarfing genes is urgently needed for the development of new high-yielding genotypes, adaptable to the warmer temperature conditions.

Acknowledgements

We thank Dr John Bailey for linguistic revisions and Dr Mihály Dernovics for his assistance in method optimisation and contribution to sample preparation for the UPLC-MS/MS analysis. Erika Gondos, Barbara Krárné-Péntek and Szilvia Fodor are acknowledged for providing technical assistance. AS and AC acknowledges funding from the Nemzeti Kutatási Fejlesztési és Innovációs Hivatal (NKFIH, NKFI-FK-124266, NKFI-129221, 2021-1.2.4-TÉT-2021-00033). TKP2021-NKTA-06 was implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021-NKTA funding scheme. TK was supported by János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00396/21/4).

    Competing interests

    None declared.

    Author contributions

    AS and AC developed the research concept and interpreted the results. AC, TK, ZG, IK and BM carried out molecular biology analyses, KÁH conducted UPLC-MS measurements, AL-T, EM, DM, FS and AS performed cytological experiments. AS wrote the paper.

    Data availability

    All data supporting the findings of this study are available within the paper and within the supplemental data published online.