The As2 Subfamily Members Of The Genetic And Functional Analysis Reveals Functional Differences Between Members

In Arabidopsis, ASYMMETIC LEAVES2 (AS2) plays an important role in leaf development. AS2 can binds to AS1, another regulater of leaf development, functioning in establishment of leaf polarity and suppression of the class I KNOX genes during leaf initiation. AS2 belongs to a large and plant-specific gene family, which contains 43 members in Arabidopsis, and is known thus far to be the only gene in the family with visible altered single mutant phenotypes. Although the AS2 function in leaf development has been characterized extensively, very little is known about functions of other members in the family.We report here a detailed genetic and functional analysis of four members that share the highest amino acid sequence similarity with AS2, including ASL1, ASL2, ASL3 and ASL4. Our results reveal that both ASL1 and ASL3 functions are partially redundant with that of AS2. Overexpression of any of these three members leads to the downregulation of KNAT1. We propose that AS2 can be grouped with ASL1 and ASL3 into a subfamily.Our results also reveal some functional divergence among the three AS2 subfamily members. For instance, plants carrying a 35S::AS2 fusion resulted in the repression of class I KNOX genes KNAT1, KNAT2 and KNAT6, whereas the 35S::ASL1 and 35S::ASL3 only repressed the expression of KNAT1/KNAT2 and KNAT1, respectively. In addition, transcript levels of the leaf polarity marker genes, PHB and KAN1, were altered in 35S::AS2 plants; however, these changes were not observed in the 35S::ASL3 plants. Furthermore, overexpression of AS2, ASL1 or ASL3 in as1 mutant background resulted in plants with different morphology, suggesting their different physical interaction with AS1. Yeast two hybrid experiment demonstrated that ASL3 but not ASL1 could interact with AS1. The functional divergence among proteins in the AS2 subfamily may depend on their structures and may reflect their authentic functions in wild type plants.To elucidate the functional domains in the AS2 subfamily proteins, we constructed chimeric proteins with different combination of AS2 and ASL3 domains, and plants carrying chimeric proteins were further analyzed. We found that the non-conserved domain of AS2 protein is important for the 35S::AS2 caused phenotypes. In addition, two motifs within the AS2 conserved domain act synergistically for the AS2 overexpression phenotypes.