Knocking-out the brassinosteroid biosynthesis genes DWARF1 or DWARF4 produces upright canopy architecture in wheat

Background: Brassinosteroids (BR) regulate multiple agronomic traits in plants, including leaf angle. BR depletion promotes an upright canopy architecture, which can improve vertical light distribution and grain yields in cereals under high planting densities. However, broad reductions in BR activity often have negative impacts on fertility and grain size. Spatially restricted modification of BR metabolism through targeting specific genes could, therefore, provide an erect canopy while minimising negative pleiotropic effects. In the BR biosynthesis pathway, DWARF1/DIMINUTO ( DWF1/DIM ) encodes a C-24 reductase that catalyses the production of the first committed sterol BR precursor, while DWF4 and DWF11 encode 22α-hydroxylase enzymes that act later in the pathway, and which exhibit partial functional redundancy. Results: Using ethyl-methanesulfonate (EMS)-induced mutations that introduce premature stop codons, we generated wheat lines lacking functional copies of all three homoeologues of DWF1 and DWF4 in the Cadenza background. The dwf4-abd mutant displayed upright leaves and a slight height reduction but showed no differences in grain size or weight when tested in glasshouse and field conditions. The dwf1-abd mutant also produced an upright canopy, but tiller number, grain size and grain weight were reduced compared to the wild-type. Scanning electron microscopy revealed that the reduced leaf angle in these mutants was due to reduced cell elongation in the auricle region of the lamina joint, causing the leaf blade to be more tightly packed around the ligule. The dwf1-abd mutant was hypersensitive to exogenous BR application. Metabolite profiling showed that concentrations of the BR precursors campesterol (CR), campestanol (CN) and 6-oxoCN were significantly reduced in dwf1-abd seedlings compared to wild-type controls, whereas levels of the bioactive BRs castasterone (CS) and brassinolide (BL) were unexpectedly elevated in both dwf1-abd and dwf4-abd . Conclusion: Wheat dwf4 null mutations confer an erect canopy architecture without yield penalties, suggesting this gene may be a promising target to develop ideotypes suitable for high-density planting. By contrast, dwf1 null mutants exhibit negative pleiotropic effects and are unlikely to be useful for agriculture. The characterised mutants advance our understanding of BR metabolism and can be explored for their potential to improve grain yields.