MicroProteins are short, single domain proteins that act by sequestering larger, multi-domain proteins into non-functional complexes. MicroProteins have been identified in plants and animals, where they are mostly involved in the regulation of developmental processes. Here we show that two Arabidopsis thaliana microProteins, miP1a and miP1b, physically interact with CONSTANS (CO) a potent regulator of flowering time. The miP1a/b-type microProteins evolved in dicotyledonous plants and have an additional carboxy-terminal PF(V/L)FL motif. This motif enables miP1a/b microProteins to interact with TOPLESS/TOPLESS-RELATED (TPL/TPR) proteins. Interaction of CO with miP1a/b/TPL causes late flowering due to a failure in the induction of FLOWERING LOCUS T (FT) expression under inductive long day conditions. Both miP1a and miP1b are expressed in vascular tissue, where CO and FT are active. Genetically, miP1a/b act upstream of CO thus our findings unravel a novel layer of flowering time regulation via microProtein-inhibition.

A defining feature of plant leaves is their flattened shape. This shape depends on an antagonism between the genes that specify adaxial (top) and abaxial (bottom) tissue identity; however, the molecular nature of this antagonism remains poorly understood. Class III homeodomain leucine zipper (HD-ZIP) transcription factors are key mediators in the regulation of adaxial-abaxial patterning. Their expression is restricted adaxially during early development by the abaxially expressed microRNA (MIR)165/166, yet the mechanism that restricts MIR165/166 expression to abaxial leaf tissues remains unknown. Here, we show that class III and class II HD-ZIP proteins act together to repress MIR165/166 via a conserved cis-element in their promoters. Organ morphology and tissue patterning in plants, therefore, depend on a bidirectional repressive circuit involving a set of miRNAs and its targets. T he morphogenesis of lateral organs in plants and animals is dependent on the specification of distinct cell types early in development. In particular, the correct patterning of adaxialabaxial tissues in plant organs such as leaves is critical for the generation of a lamina shape and the formation of a polar vascular system (1-4). Adaxial-abaxial cell-type patterning in turn depends on the restricted expression of several genes known to specify these cell types, including the class III homeodomain leucine zipper genes (HD-ZIPIIIs), KANADI genes, HD-ZIPIIs, and microRNA (MIR)165/166 (1, 2, 4-11). In general, genetic analyses have indicated that adaxial and abaxial factors act oppositely in organ patterning (1,2,4,(8)(9)(10)(11). Hence, loss-of-function mutations in genes promoting adaxial cell identity typically cause an abaxialized phenotype that correlates with the ectopic expression of abaxial genes, whereas loss-of-function mutations in abaxial genes produce an adaxialized phenotype that is accompanied by the expanded expression of adaxial genes. This antagonistic interaction between adaxial and abaxial factors may be mediated by mutually antagonistic regulation (12) or through opposing regulation of common targets (9, 13-16).A key set of transcription factors involved in plant organ polarity are the HD-ZIPIII proteins, such as REVOLUTA (REV), which specify adaxial cell fate (1, 2, 4, 17). The expression of these genes is restricted specifically to adaxial tissues via the action of two miRNA families, MIR165 and MIR166 (2, 7). In turn, the expression of these miRNAs is restricted to abaxial tissues and this restriction is essential for maintaining proper organ polarity (18).Here, we address the question of how MIR165/166 are regulated. We show that the HD-ZIPII proteins HAT3 and ATHB4 physically interact with HD-ZIPIII proteins and directly repress MIR165/166 expression via a conserved cis-element located in their promoters. This regulatory interaction largely accounts for HAT3 and ATHB4 function and reveals the molecular nature of a bidirectional repressive circuit essential to maintain balance between adaxial and abaxial tissue sp...

No abstract