UNLABELLED: The barley genome encodes a complete set of MADS-box proteins sharing homology with components of the ABCDE model, which explains the molecular basis of floral organ identity in angiosperm flowers. Although the E-class members are universally expressed across floral whorls and crucial for flower development in Arabidopsis and rice, the functional role of the barley E-class LOFSEP subfamily (comprising MADS1, MADS5, and MADS34) remains elusive, particularly during spikelet formation. Here, we characterize the single, double and triple SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-024-00182-4.