The Arabidopsis AS2 Gene Encoding a Predicted Leucine-zipper Protein Is Required for the Leaf Polarity Formation

The asymmetric leaves2 ( as2) is a classical Arabidopsis thaliana (L.) Heynh. mutant that shows leaf lobes and leaflet-like structures from the petioles of leaves. Genetic and molecular analyses have demonstrated that the AS2 function is required for repression of meristem-related homeobox genes in leaves. In this study, we describe phenotypic characterizations of new as2 alleles that are in the Landsberg erecta (Ler) genetic background. In addition to the as2 phenotypes reported previously, the new as2 mutants have some leaves with petiole growth underneath the leaf blade, showing a lotus-leaf structure. More severe rosettes leaves of the as2 mutants form a filament-like structure, reflecting a loss of the adaxial-abaxial polarity in leaves. Among as2 mutants analyzed in different genetic backgrounds, only those that are in the Ler genetic background resulted in a high frequency of the lotus-leaf structure. We have isolated the AS2 gene by map-based gene cloning. The predicted AS2 protein contains a leucine-zipper motif, and its N-terminus shares high levels of sequence similarity to those of a group of predicted proteins with no known biological functions. AS2 transcripts were detected in leaves, flowers and fruits, but absent in stems, consistent with the mutant phenotypes.

Transcription factors AS1 and AS2 interact with LHP1 to repress KNOX genes in Arabidopsis

Polycomb group proteins are important repressors of numerous genes in higher eukaryotes. However, the mechanism by which Polycomb group proteins are recruited to specific genes is poorly understood. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN 1 （LHP1）, also known as TERMINAL FLOWER 2, was originally proposed as a subunit of polycomb repressive complex 1 （PRC1） that could bind the tri-methylated lysine 27 of histone H3 （H3K27me3） established by the PRC2. In this work, we show that LHP1 mainly functions with PRC2 to establish H3K27me3, but not with PRC1 to catalyze monoubiquitination at lysine 119 of histone H2A. Our results show that complexes of the transcription factors ASYMMETRIC LEAVES 1 （AS1） and AS2 could help to establish the H3K27me3 modification at the chromatin regions of Class-I KNOTTED＇t-like homeobox （KNOX） genes BREVIPEDICELLU5 and KNAT2 via direct interactions with LHP1. Additionally, our transcriptome analysis indicated that there are probably more common target genes of AS1 and LHP1 besides Class-I KNOX genes during leaf development in Arabidopsis.

Subcellular Localizations of AS1 and AS2 Suggest Their Common and Distinct Roles in Plant Development

During leaf organogenesis, a critical step for normal leaf primordium initiation is the repression of the class 1 KNOTTED1-like homeobox （KNOX） genes. After leaf primordia are formed, they must establish polarity for normal leaf morphogenesis. Recent studies have led to the identification of a number of genes that participate in the class 1 KNOX gene repression and/or the leaf polarity establishment. ASTMMETRIC LEAVES1 and 2 （AS1 and AS2） are two of these genes, which are critical for both of these two processes. As a first step towards understanding the molecular genetic basis of the ASl-AS2 action, we determined the subcellular Iocalizations of the two proteins in both tobacco BY2 cells and Arabidopsis plants, by fusing them to yellow/cyan fluorescent protein （YFP/CFP）. Our data showed that AS1 and AS2 alone were predominantly localized in the nucleolus and the nucleoplasm, respectively. The presence of both AS1 and AS2 proteins in the same interphase cell demonstrated their co-localization in both nucleolus and nucleoplasm. In addition, AS1 alone was able to associate with the condensed chromosome in the metaphase cell. Our data suggest that AS1, AS2 and the ASl-AS2 protein complex may have distinct functions, which are all required for normal plant development.