A shade-responsive microprotein in ATHB2 regulates Arabidopsis growth and root development

Shade triggers widespread changes in transcript isoform production in Arabidopsis thaliana. Using 5’ PEAT sequencing, we identified 377 candidate microprotein-encoding transcripts arising from alternative transcription start sites, including one located within the third exon of ATHB2, a class II HD-ZIP transcription factor central to shade avoidance. The encoded microprotein, ATHB2miP, lacks the DNA-binding homeodomain but retains the leucine zipper domain, through which it forms heterodimers with full-length ATHB2, inhibits its DNA-binding activity, and disrupts its localization to nuclear photobodies. Ectopic expression of ATHB2miP phenocopies loss of ATHB2, deregulating genes involved in auxin signaling, root development, and iron homeostasis. Complementation of the athb2 mutant with a construct carrying a silent mutation at the ATHB2miP start codon results in exaggerated hypocotyl elongation under both white light and shade, demonstrating that ATHB2miP is required to restrain excessive elongation growth. Immunoprecipitation mass spectrometry confirmed that this mutation redirects translation to alternative initiation sites, producing distinct proteoforms absent from wild-type plants. Additionally, ATHB2 suppresses iron uptake genes, and iron availability modulates shade growth responses in an ATHB2-dependent manner. Together, these findings establish ATHB2miP as a regulator of an ATHB2 feedback circuit that integrates light and nutritional signals to coordinate shoot elongation and root development.