Elongated outer glume characterizes polish wheat (Triticum polonicum) as a unique tetraploid wheat species. The genetic control of long-glume trait by a single semi-dominant P1 (from Polish wheat) locus has been established over 100 years ago, whereas its causal gene and the precise molecular details remain elusive.

Based on map-based cloning, we isolate VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT-A2), an SVP-clade MADS-box transcription factor (TF) coding gene, as P1 candidate. Genetic evidence suggests that in T. polonicum, a naturally occurred sequence rearrangement in the intron-1 region of VRT-A2 leads to its ectopic expression in floral organs, whereby the long-glume phenotype appears.

More interestingly, we demonstrate that the intron-1 region is a key ON/OFF molecular switch for VRT-A2 expression, due to its sequence features for not only recruiting transcriptional repressors, but also conferring intron-mediated transcriptional enhancement. Genotypic analyses using wheat accessions indicate that P1 locus is likely derived from a single natural mutation in tetraploid wheat, which later has been inherited by hexaploid T. petropavlovskyi.

It should be noted that overdose activation of VRT-A2 significantly enhances grain weight but also triggers increased rudimentary basal spikelets and decreased grain number per spike. This yield penalty has presented challenge for the use of VRT-A2 in high-yield wheat breeding practice.

Aiming to decouple the pleiotropic effects of VRT-A2 on different tissues, we further make efforts to characterize the upstream regulatory modules for fine-tuning VRT-A2 expression, and confirm that: an AP2/ERF-type transcription factor multi-floret spikelet1 (TaMFS1) represses VRT-A2 expression by recruiting transcriptional co-repressor and histone deacetylase; while a structure-specific recognition protein TaSSRP1 facilitates VRT-A2 activation by assembling Mediator and RNA polymerase.

By deleting TaMFS1, we enable a moderate upregulation of VRT-A2 expression, resulting in significant increase of grain weight without yield penalty. Our study provides a feasible strategy for overcoming the trade-offs of pleotropic genes by editing their upstream transcriptional regulators.