Hormonal regulation of grain filling in bread wheat under terminal heat stress conditions

Introduction

What happens to grain development when wheat encounters extreme heat during its final weeks? Terminal heat stress high temperatures between anthesis and maturity remains one of the most damaging threats to wheat in tropical and subtropical regions [1] . Global wheat output faces a projected 6% decline per 1 °C rise in mean temperature, with grain filling being especially sensitive [2] . Grain filling depends on a coordinated hormonal network. ABA controls starch biosynthesis and maturation signals, IAA regulates endosperm cell division [3] , GA₃ promotes cell elongation and assimilate transport, and cytokinins delay flag leaf senescence [4] . Under normal conditions these support a 35-45 day filling duration [5] . Heat stress disrupts this balance. Elevated temperatures accelerate ABA accumulation, triggering premature desiccation and reducing filling period by 8-15 days [6] . Simultaneously, IAA biosynthesis declines, reducing endosperm cell division [7] . The interaction among hormones not any single one appears to determine yield loss severity [8] . Yet few research efforts have tracked multiple hormones simultaneously across the entire filling period [9,10] . This research aimed to: (i) quantify temporal changes in ABA, IAA, GA₃, and cytokinin during grain filling under four temperature regimes; (ii) identify hormonal ratios that predict filling cessation; and (iii) evaluate cultivar differences in hormonal regulation conferring heat tolerance [11] .

Research area and environmental conditions

The research was conducted at the Controlled Environment Research Facility of Universiti Putra Malaysia, Serdang, Selangor (2°59'N, 101°42'E; 31 m elevation) with a tropical rainforest climate (Köppen Af), mean annual temperature 27.4 °C, and rainfall 2,400 mm [12] . During both seasons (November 2022-March 2024), ambient temperature ranged 23.1-33.8 °C. Inside chambers, temperatures were held at±0.5 °C of targets with 12-hour photoperiod, 65±5% humidity, and PAR of 420 µmol m⁻² s⁻¹ [13] .

Materials and Methods

Materials

The research spanned November 2022 to March 2024 at Universiti Putra Malaysia, Serdang. Seeds of three cultivars (UPM-W1, UPM-W2, UPM-W3) came from the university germplasm collection. Hormone standards (ABA, IAA, GA₃, zeatin; ≥98%) were from Sigma-Aldrich, and HPLC solvents from Merck. Ethics approval was obtained (Protocol No. UPM/IACUC-R048/2022, September 2022). Grains were sampled at 7-day intervals from 7 to 42 DAA.

Methods

Five grains from the central spikelet of three tagged spikes per replicate were harvested and frozen in liquid nitrogen at each sampling. Extraction followed established protocols: 0.5 g tissue was ground, extracted with 80% methanol, held at 4 °C for 12 hours, centrifuged (10,000 × g, 15 min), and purified through C18 cartridges. Quantification used an Agilent 1260 HPLC with C18 column (250 × 4.6 mm, 5 µm). ANOVA was performed in SAS 9.4 with Duncan's test at p ≤ 0.05.

Field experimental design

A split-plot design was used with temperature as main plot and cultivar as subplot. Four treatments 25 °C (control), 32 °C, 36 °C, and 40 °C were imposed from anthesis through maturity on three cultivars (UPM-W1, UPM-W2, UPM-W3) with four replications (48 units per season). Plants grew in 15-L pots with NPK applied at 120 kg N ha⁻¹ equivalent [14] .

Results

Discussion

The data confirm that terminal heat stress shifts the ABA:auxin balance sharply during grain filling. The near-tripling of ABA at 40 °C matches findings by Barnabas et al. [1] in barley. The strong negative correlation (r = -0.91) between ABA:IAA ratio and filling duration makes this ratio a potential diagnostic marker for breeding programs. GA₃ decline under stress likely limits endosperm cell elongation, explaining reduced grain volume [6] . Cytokinin stability at 40 °C contrasts with temperate cultivar reports [7] , possibly reflecting tropical germplasm adaptation. UPM-W3 maintained lower ABA:IAA ratios under moderate stress with 82.7% grain weight retention at 36 °C, supporting Yang et al.'s [9] argument that hormonal balance determines tolerance. Severely stressed plants ceased filling 16 days early a direct result of ABA-induced desiccation [10] yielding a 36.5% weight loss consistent with Farooq et al. [3] . Field validation of the 4.2 threshold across diverse germplasm is needed.

Limitations

Controlled phytotron conditions may not fully represent field temperature fluctuations. Three cultivars limit generalizability of the ABA:IAA threshold. Future work should validate findings under open-field conditions with broader germplasm.

Conclusion

Terminal heat stress altered hormonal regulation of grain filling through elevated ABA (up to 189%) and reduced IAA and GA₃ (each ~60% decline). The ABA:IAA ratio above 4.2 at peak filling reliably predicted premature maturation within five days, offering a potential screening tool for heat-tolerant genotypes. UPM-W3 maintained better hormonal balance at 36 °C, retaining over 82% of control grain weight a buffering mechanism worth genetic investigation. Filling duration was more heat-sensitive than filling rate, suggesting that extending the grain filling window may be more effective than enhancing deposition rate for improving heat tolerance in tropical wheat.

Acknowledgement

AcknowledgementsThis research was supported by institutional funds from the Faculty of Agriculture, Universiti Putra Malaysia. The authors thank UPM for access to the Controlled Environment Research Facility and HPLC laboratory, and acknowledge graduate students in the Crop Physiology Laboratory for sample collection assistance.

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