EXPANSIN A6 and GRAIN WIDTH 2 genes differentially affect grain weight, grain number and yield of wheat through their physiological determinants.

The physiological basis of the trade-off between grain number (GN) and thousand grain weight (TGW) is key to wheat yield improvement. To that end, three wheat line groups were assessed at conventional (300-350 pl m-2) and low (44 pl m-2) planting rates in field experiments: TaExpA6 (EXPANSIN A6 overexpression), TaGW2 (GRAIN WIDTH 2 triple knockout), and TaP1xGW2A (VEGETATIVE TO REPRODUCTIVE TRANSITION 2 ectopic expression, and TaGW2-A knockout), together with their wild types (WT). Lines TaExpA6 and TaGW2 increased TGW over their respective WTs, whereas line TaP1xGW2A showed no effect on this trait. The transgenic TaExpA6 line was the only one to achieve greater grain yield (GY) than its WT, as the TGW increase conferred by the TaGW2 triple mutant was fully offset by reductions in spike number (SpN) and grain number per spike (GNS). Contrasting effects of TaExpA6 and TaGW2 lines were identified on the ovary weight and floret dynamics, likely associated with the trade-off between TGW and GN. The TaExpA6 construct drives targeted expression in developing grain tissues at post-anthesis, avoiding the overlap with GN determination. In contrast, TaGW2 disruption constrains tillering, likely as a pleiotropic effect, and shifts intra-spike resource allocation, promoting early ovary growth at the expense of distal floret development and GNS. Low planting rate increased individual spike dry weight at anthesis across all line groups, mitigating the TGW-GNS trade-off and increasing both traits simultaneously. These results highlight that post-anthesis, grain-specific gene expression plays an important role in mitigating the trade-off between TGW and GN.