A genetic link between epigenetic repressor AS1–AS2 and DNA replication factors in establishment of adaxial–abaxial leaf polarity of <i>Arabidopsis</i>

Abstract

Balanced development of adaxial and abaxial domains in leaf primordia is critical for the formation of flat symmetric leaf lamina. Arabidopsis ASYMMETRIC LEAVES1 (AS1) and AS2 proteins form a complex (AS1-AS2), which acts as key regulators for the adaxial development by the direct repression of expression of the abaxial gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). Many modifier mutations have been identified, which enhance the defect of as1 and as2 mutations to generate abaxialized filamentous leaves without adaxial traits, suggesting that the development of the adaxial domain is achieved by cooperative repression by AS1-AS2 and the wild-type proteins corresponding to the modifiers. Mutations of several genes for DNA replication-related chromatin remodeling factors such as Chromatin Assembly Factor-1 (CAF-1) have been also identified as modifiers. It is still unknown, however, whether mutations in genes involved in DNA replication themselves might act as modifiers. Here we report that as1 and as2 mutants grown in the presence of hydroxyurea, a known inhibitor of DNA replication, form abaxialized filamentous leaves in a concentration-dependent manner. We further show that a mutation of the INCURVATA2 (ICU2) gene, which encodes the putative catalytic subunit of DNA polymerase α, and a mutation of the Replication Factor C Subunit3 (RFC3) gene, which encodes a protein used in replication as a clamp loader, act as modifiers. In addition, as2-1 icu2-1 double mutants showed increased mRNA levels of the genes for leaf abaxialization. These results suggest a tight link between DNA replication and the function of AS1-AS2 in the development of flat leaves.

Patterning a Leaf by Establishing Polarities

“…Two nucleolus localized proteins, NUCLEOLINI and RNA HELICASE10 are associated with the AS2-dependent DNA methylation of ETT (Vial-Pradel et al, 2018). It is also reported that replication factor C subunit 3 and DNA polymerase subunit INCURVATA2 function as modifiers of AS2 in repressing ETT and ARF4 (Luong et al, 2018). Therefore, a feedback loop among ETT-KAN-AS2 acts to spatially define adaxial-abaxial cell fates.…”

Arabidopsis ASYMMETRIC LEAVES2 (AS2): roles in plant morphogenesis, cell division, and pathogenesis

“…Similar abnormalities are observed in leaf lamina of the as1 mutant leaves as well (Candela et al 1999 ; Tsukaya and Uchimiya 1997 ), suggesting that AS2 and AS1 are involved in similar processes to develop flat symmetric and extended leaves and vascular systems consisting of the prominent midvein and complex fine networks of leaf venation. Many mutations that enhance phenotypes of as2 and as1 have been identified (Horiguchi et al 2011 ; Ishibashi et al 2012 ; Kojima et al 2011 ; Luong et al 2018 ; Matsumura et al 2016 ; Pinon et al 2008 ; Ueno et al 2007 ; Yao et al 2008 ), and see additional references summarized by Machida et al ( 2015 ). We designated the causative genes as “modifiers” for as2 and as1 mutants.…”

“…Chemicals that enhance the phenotypes of as2 and as1 single mutants have been also identified. They include two specific inhibiters against histone deacetylases, trichostatin A (TSA) and 4-(dimethylamino)-N-[6-(hydroxyamino)-6-oxohexyl]-benzamide (CAY) (Ueno et al 2007 ); berberine (a benzylisoquinoline alkaloid), which acts to bind guanine-rich triplex or quadruplex DNA and possesses various biological activities, as well as has been used in medicines (Nakagawa et al 2012 ); and hydroxyurea, a known DNA replication inhibitor (Luong et al 2018 ). In addition, by screening for natural compounds that stimulate the formation of filamentous leaves in the as2 background, we have found more compounds that act as modifier mutations (our unpublished observations).…”

Molecular and Hormonal Regulation of Leaf Morphogenesis in Arabidopsis

“…The direct interaction between KAN and ARF indicates that overlapping patterns control the polarity of the leaf. Both the ETT and ARF4 , as a target of TAS3, are derived from trans-acting siRNAs (ta-siRNAs) that up-regulate small interfering RNA (siRNA) [ 98 , 176 ]. According to this experiment, ta-siRNA insensitive ETT or ETT overexpression in the rdr6-15 mutant background showed a defect in leaf morphology.…”