Regulation of MIR165/166 by class II and class III homeodomain leucine zipper proteins establishes leaf polarity

Abstract

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...

Multiple Pathways in the Control of the Shade Avoidance Response

“…Unfortunately, multiple loss-of-function HD-Zip II mutants are difficult to test in shade, since they are strongly altered in embryo, SAM activity, leaf polarity, gynoecium and fruit development under simulated sunlight conditions [121,136,138], implying a fundamental role of these proteins in the regulation of plant growth and development. Indeed, there are evidences that alteration of selected HD-Zip II proteins affects at least a regulatory circuit between HD-Zip II and HD-Zip III transcription factors [111,117,121,136,155] and hormones signal transduction pathways [139,180].…”

“…Considering that HD-Zip II proteins work as repressors of gene expression [27,121,123], it was proposed that, among other possibilities, they may negatively regulate molecules that restrict HD-Zip III expression [112]. Interestingly, it was recently shown that REV, whose expression is restricted to the adaxial side of the leaf through the activity of miR165/166, physically interacts with HAT3 and ATHB4 to directly repress MIR165/166 expression in the leaf adaxial domain [155].…”

Cooperative Regulatory Functions of miR858 and MYB83 during Cyst Nematode Parasitism

“…A reciprocal feedback loop controlling the expression of miR396 and its target transcription factors GRF1 and GRF3 has been demonstrated to coordinate transcriptional events required for proper syncytium formation and function Liu et al, 2014b). In addition, a number of miRNAs and their transacting targets were found to be intricately connected through feedback circuits in different growth and developmental contexts, where robust and adaptable transcriptional responses were established (Xie et al, 2003;Gutierrez et al, 2009;Wu et al, 2009;Marin et al, 2010;Yant et al, 2010;Merelo et al, 2016). Our data suggest that the miR858/MYB83 regulatory circuit may involve MYB12, a confirmed target of miR858, whose transcript abundance was positively regulated by MYB83.…”

Getting leaves into shape: a molecular, cellular, environmental and evolutionary view

“…At the heart of this complex network lie class III HD-ZIP transcription factors, such as PHABULOSA, PHAVOLUTA and REVOLUTA, and the GARP family transcription factors KANADI 1-4 (KAN1-4), which determine adaxial and abaxial identities, respectively Kerstetter et al, 2001;McConnell et al, 2001;Emery et al, 2003). In addition, class II HD-ZIP proteins and the AS1/AS2 complex determine adaxial identity (Husbands et al, 2015;Merelo et al, 2016), whereas the auxin response factors ARF2, ARF3 (also known as ETTIN) and ARF4 contribute to abaxial identity (Pekker et al, 2005;Guan et al, 2017). The expression patterns of the adaxial and abaxial determinants resolve into complementary domains as a result of different regulatory mechanisms.…”

“…For instance, KAN1 directly represses AS2 (Wu et al, 2008) and, in turn, the AS1-AS2 complex represses the abaxial factors MIR166 and ARF3 (Husbands et al, 2015). In parrallel, class III HD-ZIP genes are repressed by miR165/166, and the genes coding for these miRNAs are repressed by a class II-class III HD-ZIP protein complex (Merelo et al, 2016). Finally, auxin also plays a role in establishing polarity: its depletion from the adaxial domain, which results from auxin flow from the young leaf primordium to the meristem, is proposed to establish a transient low auxin domain that contributes to leaf polarity (Qi et al, 2014).…”