KANADI1 regulates adaxial–abaxial polarity in Arabidopsis by directly repressing the transcription of ASYMMETRIC LEAVES2

Abstract

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...

SWP73 Subunits of Arabidopsis SWI/SNF Chromatin Remodeling Complexes Play Distinct Roles in Leaf and Flower Development

“…We observed that transcript levels of KAN1, AS1, and AS2 were simultaneously increased 1.8-to 2.0-fold in swp73b leaves compared with the wild type ( Figure 4B). As KAN1 is a transcription repressor of AS2 and vice versa (Wu et al, 2008), this observation indicated that both abaxial and adaxial repressor activities were probably compromised in the swp73b mutant. Elevated KAN1 levels are known to result in feedback inhibition of KAN2, KAN3, and YAB5, which are involved in abaxial cell fate specification (Merelo et al, 2013;Huang et al, 2014).…”

The ASYMMETRIC LEAVES Complex Employs Multiple Modes of Regulation to Affect Adaxial-Abaxial Patterning and Leaf Complexity

“…Additionally, repression of TAS3A may result from cooperativity between ARF3/ARF4 and factors that are misexpressed only in as2. One such candidate is KAN1, which physically interacts with ARF3 (Kelley et al, 2012), and whose expression extends into the adaxial side of as2 leaves (Wu et al, 2008). The fact that TAS3A expression persists in as2 seems to favor the requirement of a cofactor during the ARF3/ARF4-mediated repression of TAS3A.…”

“…KAN proteins directly repress AS2 expression (Wu et al, 2008), making them likely candidates to set up this polarized activity. Indeed, KAN dynamics appear complementary to AS2, with transcripts accumulating on the abaxial side of developing leaves (Kerstetter et al, 2001;Wu et al, 2008).…”

Auxin depletion from leaf primordia contributes to organ patterning

“…Regulatory genes expressed in the abaxial domain suppress those expressed in the adaxial domain and vice versa. MicroRNAs 165 and 166 (MiR165/166) and transcription factor-encoding KANADI (KAN) genes are expressed in the abaxial domain and restrict the expression of class III homeodomain leucine zipper (HD-ZIPIII) genes PHABULOSA, PHAVOLUTA, and REVOLUTA (REV) to the adaxial domain (7,8), and KANs directly repress LOB-domain gene ASYM-METRIC LEAVES2 (AS2), another adaxial regulator (9). The trans-acting short interfering RNA3 (TAS3), another small RNA, is expressed on the adaxial side to restrict their targets, ARFdomain genes ETTIN (ETT, also known as ARF3) and ARF4, to the abaxial domain (10)(11)(12).…”