Developmental dynamics of transcription initiation highlight promoter shifts as a multifaceted regulatory mechanism in barley

Alternative promoter usage is a widespread regulatory mechanism whereby a single gene utilizes two or more transcription start sites (TSSs), enabling the production of functionally distinct transcript and protein isoforms in a tissue-, developmental stage-, or condition-specific manner. While this phenomenon has been extensively studied in metazoans, genome-wide characterization of alternative promoter usage in crop species is still lacking. Here, we use Cap Analysis of Gene Expression (CAGE) to characterize genome-wide TSS dynamics across three stages of barley embryo development and the developing inflorescence. We identify 2,129 developmental promoter shifts relating to 1,353 genes, corresponding to ~5% of genes expressed in at least one of the four developmental stages. One-third of these shifts coincided with significant differential gene expression, and the affected genes were enriched for developmental and regulatory functions, particularly those related to cell wall biosynthesis and hormone signaling. Furthermore, for 521 genes, we predict potential effects of alternative TSS usage on translational efficiency or protein structure, suggesting regulatory consequences that extend beyond changes in transcript abundance. Together, our findings suggest that alternative promoter selection is an important regulatory mechanism during barley development and provide new insights into the developmental regulation of gene expression in cereals. Moreover, we provide a gene-level catalog of developmental promoter shifts in barley, offering a valuable resource for promoter editing and engineering to fine-tune gene expression for desirable agronomic traits.