Lateral organ polarity in Arabidopsis is regulated by antagonistic interactions between genes that promote either adaxial or abaxial identity, but the molecular basis of this interaction is largely unknown. We show that the adaxial regulator ASYMMETRIC LEAVES2 (AS2) is a direct target of the abaxial regulator KANADI1 (KAN1), and that KAN1 represses the transcription of AS2 in abaxial cells. Mutation of a single nucleotide in a KAN1 binding site in the AS2 promoter causes AS2 to be ectopically expressed in abaxial cells, resulting in a dominant, adaxialized phenotype. We also show that the abaxial expression of KAN1 is mediated directly or indirectly by AS2. These results demonstrate that KAN1 acts as a transcriptional repressor and that mutually repressive interactions between KAN1 and AS2 contribute to the establishment of adaxial-abaxial polarity in plants.A daxial-abaxial polarity in plants is specified by interactions between genes that individually specify either adaxial or abaxial identity (1, 2). In Arabidopsis, adaxial identity is specified by class III homeodomain leucine zipper (HD-ZIPIII) transcription factors (3, 4), the transcription factor ASYMMETRIC LEAVES2 (AS2) (5, 6), and the transacting siRNA tasiARF (7), whereas abaxial identity is specified by KANADI (KAN) (8, 9), YABBY (YAB) (10, 11), AUXIN RESPONSE FACTOR (ARF) (12), and LITTLE ZIPPER (ZPR) (13) transcription factors and by the miRNAs miR165/miR166 (14-16). Genetic analysis indicates that many of these genes interact antagonistically: loss-of-function mutations in adaxial genes typically produce an abaxialized phenotype that is accompanied by the expanded expression of abaxial genes, whereas loss-of-function mutations in abaxial genes produce an adaxialized phenotype that is associated with the expanded expression of adaxial genes; mutations or transgenes that produce ubiquitous expression of these genes have a phenotype opposite to that of the loss-offunction mutations. miR165/166 and tasiARF repress the expression of their targets, respectively, HD-ZIPIII genes and ARF3/ETTIN, by directing the cleavage of the transcripts of these genes (14, 17). The molecular basis for the antagonistic interactions between other adaxial and abaxial regulators is unknown.KAN1, KAN2, and KAN3 are members of the GARP family of transcription factors and act redundantly to promote abaxial identity in both lateral organs and the shoot axis. These genes are expressed in abaxial cells of lateral organs and peripheral cells of the hypocotyl and stem (9, 16). Loss-of-function mutations in individual genes have relatively weak effects on organ polarity (9, 18), but kan1 kan2 (8) and kan1 kan2 kan3 (16) mutants are strongly adaxialized and resemble plants ectopically expressing the HD-ZIPIII genes PHB, PHV, and REV (3,15) or the LOB gene AS2 (5, 6). Consistent with this observation, PHB is abaxially expressed in kan1 kan2 kan3 triple mutants (16). The effect of kan on the expression of AS2 has not been examined.Here, we show that KAN1 promotes abaxial identity by di...

The formation of leaves and other lateral organs in plants depends on the proper specification of adaxial-abaxial (upper-lower) polarity. KANADI1 (KAN1), a member of the GARP family of transcription factors, is a key regulator of abaxial identity, leaf growth, and meristem formation in Arabidopsis thaliana. Here, we demonstrate that the Myb-like domain in KAN1 binds the 6-bp motif GNATA(A/T) and that this motif alone is sufficient to squelch transcription of a linked reporter in vivo. In addition, we report that KAN1 acts as a transcriptional repressor. Among its targets are genes involved in auxin biosynthesis, auxin transport, and auxin response. Furthermore, we find that the adaxializing HD-ZIPIII transcription factor REVOLUTA has opposing effects on multiple components of the auxin pathway. We hypothesize that HD-ZIPIII and KANADI transcription factors pattern auxin accumulation and responsiveness in the embryo. Specifically, we propose the opposing actions of KANADI and HD-ZIPIII factors on cotyledon formation (KANADI represses and HD-ZIPIII promotes cotyledon formation) occur through their opposing actions on genes acting at multiple steps in the auxin pathway.

The broadly conserved Class III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) and KANADI transcription factors have opposing and transformational effects on polarity and growth in all tissues and stages of the plant's life. To obtain a comprehensive understanding of how these factors work, we have identified transcripts that change in response to induced HD-ZIPIII or KANADI function. Additional criteria used to identify high-confidence targets among this set were presence of an adjacent HD-ZIPIII binding site, expression enriched within a subdomain of the shoot apical meristem, mutant phenotype showing defect in polar leaf and/or meristem development, physical interaction between target gene product and HD-ZIPIII protein, opposite regulation by HD-ZIPIII and KANADI, and evolutionary conservation of the regulator-target relationship. We find that HD-ZIPIII and KANADI regulate tissue-specific transcription factors involved in subsidiary developmental decisions, nearly all major hormone pathways, and new actors (such as INDETERMINATE DOMAIN4) in the ad/abaxial regulatory network. Multiple feedback loops regulating HD-ZIPIII and KANADI are identified, as are mechanisms through which HD-ZIPIII and KANADI oppose each other. This work lays the foundation needed to understand the components, structure, and workings of the ad/abaxial regulatory network directing basic plant growth and development.

The Arabidopsis petal is a simple laminar organ whose development is largely impervious to environmental effects, making it an excellent model for dissecting the regulation of cell-cycle progression and post-mitotic cell expansion that together sculpt organ form. Arabidopsis petals grow via basipetal waves of cell division, followed by a phase of cell expansion. RABBIT EARS (RBE) encodes a C2H2 zinc finger transcriptional repressor and is required for petal development. During the early phase of petal initiation, RBE regulates a microRNA164-dependent pathway that controls cell proliferation at the petal primordium boundaries. The effects of rbe mutations on petal lamina growth suggest that RBE is also required to regulate later developmental events during petal organogenesis. Here, we demonstrate that, early in petal development, RBE represses the transcription of a suite of CIN-TCP genes that in turn act to inhibit the number and duration of cell divisions; the temporal alleviation of that repression results in the transition from cell division to post-mitotic cell expansion and concomitant petal maturation.

The Arabidopsis thaliana MADS box transcription factors APETALA3 (AP3) and PISTILLATA (PI) heterodimerize and are required to specify petal identity, yet many details of how this regulatory process is effected are unclear. We have identified three related genes, BHLH136/BANQUO1 (BNQ1), BHLH134/BANQUO2 (BNQ2), and BHLH161/BANQUO3 (BNQ3), as being directly and negatively regulated by AP3 and PI in petals. BNQ1, BNQ2, and BNQ3 encode products belonging to a family of atypical non-DNA binding basic helix-loop-helix (bHLH) proteins that heterodimerize with and negatively regulate bHLH transcription factors. We show that bnq3 mutants have pale-green sepals and carpels and decreased chlorophyll levels, suggesting that BNQ3 has a role in regulating light responses. The ap3 bnq3 double mutant displays pale second-whorl organs, supporting the hypothesis that BNQ3 is downstream of AP3. Consistent with a role in light response, we show that the BNQ gene products regulate the function of HFR1 (for LONG HYPOCOTYL IN FAR-RED1), which encodes a bHLH protein that regulates photomorphogenesis through modulating phytochrome and cryptochrome signaling. The BNQ genes also are required for appropriate regulation of flowering time. Our results suggest that petal identity is specified in part through downregulation of BNQ-dependent photomorphogenic and developmental signaling pathways.

Genetic stocks and growth conditionsArabidopsis thaliana plants were grown at 22°C under 16-hour light/8-hour dark conditions. The rbe-2 (Salk_037010), rbe-3, mir164b-1 (Salk_136105), cuc1-1 and cuc2-1 seeds were obtained from the Arabidopsis Biology Resource Center (ABRC). mir164a-4 (GABI SUMMARYThe establishment and maintenance of organ boundaries are vital for animal and plant development. In the Arabidopsis flower, three microRNA164 genes (MIR164a, b and c) regulate the expression of CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, which encode key transcriptional regulators involved in organ boundary specification. These three miR164 genes are expressed in distinct spatial and temporal domains that are crucial for their function. Here, we show that the C2H2 zinc finger transcriptional repressor encoded by RABBIT EARS (RBE) regulates the expression of all three miR164 genes. Furthermore, we demonstrate that RBE directly interacts with the promoter of MIR164c and negatively regulates its expression. We also show that the role of RBE in sepal and petal development is mediated in part through the concomitant regulation of the CUC1 and CUC2 gene products. These results indicate that one role of RBE is to fine-tune miR164 expression to regulate the CUC1 and CUC2 effector genes, which, in turn, regulate developmental events required for sepal and petal organogenesis.KEY WORDS: Arabidopsis, miRNA, Flower development RBE controls microRNA164 expression to effect floral organogenesis

One of the biggest unanswered questions in developmental biology is how growth is controlled. Petals are an excellent organ system for investigating growth control in plants: petals are dispensable, have a simple structure, and are largely refractory to environmental perturbations that can alter their size and shape. In recent studies, a number of genes controlling petal growth have been identified. The overall picture of how such genes function in petal organogenesis is beginning to be elucidated. This review will focus on studies using petals as a model system to explore the underlying gene networks that control organ initiation, growth, and final organ morphology.

MicroRNAs (miRNAs) are a group of endogenous noncoding small RNAs frequently 21 nucleotides long. miRNAs act as negative regulators of their target genes through sequence-specific mRNA cleavage, translational repression, or chromatin modifications. Alterations of the expression of a miRNA or its targets often result in a variety of morphological and physiological abnormalities, suggesting the strong impact of miRNAs on plant development. Here, we review the recent advances on the functional studies of plant miRNAs. We will summarize the regulatory networks of miRNAs in a series of developmental processes, including meristem development, establishment of lateral organ polarity and boundaries, vegetative and reproductive organ growth, etc. We will also conclude the conserved and species-specific roles of plant miRNAs in evolution and discuss the strategies for further elucidating the functional mechanisms of miRNAs during plant development.