Background: Drought is a major environmental stress that affects crop productivity worldwide. Although previous research demonstrated links between strigolactones (SLs) and drought, here we used barley (Hordeum vulgare) SL-insensitive mutant hvd14 (dwarf14) to scrutinize the SL-dependent mechanisms associated with water deficit response. Results: We have employed a combination of transcriptomics, proteomics, phytohormonomics analyses, and physiological data to unravel differences between wild-type and hvd14 plants under drought. Our research revealed that drought sensitivity of hvd14 is related to weaker induction of abscisic acid-responsive genes/proteins, lower jasmonic acid content, higher reactive oxygen species content, and lower wax biosynthetic and deposition mechanisms than wild-type plants. In addition, we identified a set of transcription factors (TFs) that are exclusively drought-induced in the wild-type barley. Conclusions: Critically, we resolved a comprehensive series of interactions between the drought-induced barley transcriptome and proteome responses, allowing us to understand the profound effects of SLs in alleviating water-limiting conditions. Several new avenues have opened for developing barley more resilient to drought through the information provided. Moreover, our study contributes to a better understanding of the complex interplay between genes, proteins, and hormones in response to drought, and underscores the importance of a multidisciplinary approach to studying plant stress response mechanisms.

SUMMARY: The distinctive spike‐shaped inflorescence architecture in temperate cereals is governed by a complex interplay of genetic factors, including the orthologous WHEAT FRIZZY PANICLE ( WFZP ) and barley COMPOSITUM 2 ( COM2 ), whose products belong to the APETALA 2/ ETHYLENE RESPONSE FACTOR (AP2/ERF) family of transcription factors. However, the precise regulatory mechanism driving their function as well as the influence of allele dosage on spike formation and fertility remain to be elucidated specifically in wheat. A comprehensive analysis of the phenotype and gene function was conducted to unravel the genetic and molecular mechanisms underlying the peculiar rattail 1 ( rtt1 ) barley phenotype. In wheat, new WFZP alleles were generated, including knockouts and in‐frame mutations in all three homoeoalleles. Our genetic analysis revealed that the rtt1 phenotype is caused by a conserved COM2 mutation. The subsequent analysis of the transcriptome data elucidated the molecular mechanisms of rtt1 for spikelet meristem identity, underscored by a predicted compromised DNA‐binding capacity of rtt1. The combination of our newly generated mutant alleles in wheat has revealed the intricate regulation of inflorescence, grain, and root development, which is controlled by WFZP. This provides evidence of the indispensable role of functional WFZP proteins, with a particular focus on the WFZP‐B allele, which has hitherto received limited attention. This study also demonstrates the allele‐specific impact of WFZP on root development, influencing branching and length. The findings of this study demonstrate that COM2 and WFZP exert dosage‐dependent regulatory influence on the inflorescence and root architecture in Triticeae cereals.