Dual regulation of ETTIN (ARF3) gene expression by AS1-AS2, which maintains the DNA methylation level, is involved in stabilization of leaf adaxial-abaxial partitioning in Arabidopsis

Abstract

Leaf primordia are generated at the periphery of the shoot apex, developing into flat symmetric organs with adaxial-abaxial polarity, in which the indeterminate state is repressed. Despite the crucial role of the ASYMMETRIC LEAVES1 (AS1)-AS2 nuclear-protein complex in leaf adaxial-abaxial polarity specification, information on mechanisms controlling their downstream genes has remained elusive. We systematically analyzed transcripts by microarray and chromatin immunoprecipitation assays and performed genetic rescue of as1 and as2 phenotypic abnormalities, which identified a new target gene, ETTIN (ETT)/AUXIN RESPONSE FACTOR3 (ARF3), which encodes an abaxial factor acting downstream of the AS1-AS2 complex. While the AS1-AS2 complex represses ETT by direct binding of AS1 to the ETT promoter, it also indirectly activates miR390- and RDR6-dependent post-transcriptional gene silencing to negatively regulate both ETT and ARF4 activities. Furthermore, AS1-AS2 maintains the status of DNA methylation in the ETT coding region. In agreement, filamentous leaves formed in as1 and as2 plants treated with a DNA methylation inhibitor were rescued by loss of ETT and ARF4 activities. We suggest that negative transcriptional, post-transcriptional and epigenetic regulation of the ARFs by AS1-AS2 is important for stabilizing early leaf partitioning into abaxial and adaxial domains.

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

“…Furthermore, consistent with data showing AS1 and AS2 operate as a complex (Lin et al, 2003;Guo et al, 2008), ChIP assays on seedlings expressing a functional AS1-HA fusion showed specific localization of AS1 to these ARF3, MIR166A, YAB5, and TAS3A promoter regions (Supplemental Figure 1E). Importantly, while a recent genome-wide analysis of AS1 activity identified ARF3 as a potential direct target (Iwasaki et al, 2013), the binding site proposed to mediate this regulation is distinct from the AS1-AS2 target site defined here. Moreover, the presence and position of the AS1-AS2 binding site identified in this study neatly explains the observation that deletion of the proposed AS1 target site from an ARF3:GUS transcriptional reporter does not phenocopy its pattern of misexpression in as1.…”

The Sussex signal: insights into leaf dorsiventrality

“…Moreover, while the function of other polarity determinants in promoting either adaxial or abaxial fate is conserved, their input into the regulation of YABBY genes must have diverged during plant evolution. Many more targets acting downstream of the core polarity network have recently been identified in Arabidopsis (Reinhart et al, 2013;Merelo et al, 2013;Iwasaki et al, 2013;Huang et al, 2014;Xie et al, 2015) and it will be interesting to see the extent to which these are conserved.…”

STENOFOLIA Recruits TOPLESS to RepressASYMMETRIC LEAVES2at the Leaf Margin and Promote Leaf Blade Outgrowth inMedicago truncatula

“…AS2 forms a repressive complex with AS1 and prevents meristematic activity in leaves by directly repressing the transcription of KNOTTED1-like homeobox (KNOX) genes (Guo et al, 2008;Luo et al, 2012). The AS1/AS2 complex has also recently been shown to directly or indirectly repress the abaxial factors ETTIN/AUXIN RESPONSE FACTOR3 (ARF3) and ARF4 (Iwasaki et al, 2013). The repression of KNOX genes in leaves by the AS1/AS2 complex is required not only for proper differentiation of leaf cells but also for the commitment of leaf founder initial cells to form a leaf primordium, where the downregulation KNOX genes at the incipient primordium site is required for leaf initiation (Hake et al, 2004;Moon and Hake, 2011).…”