| To identify other factors invovled in AS1-AS2 pathway, we carried out screening for genetic enhancers of as1/as2 by EMS mutagenesis. During screening, we recovered a mutant, designated dm1, with dramatically enhanced as1/as2 phenotypes. Genetic characterizations of the dm1 mutant resulted in the discovery of a novel single mutant, named ae1 for asymmetric leaves1/2 enhancer. Through SEM (Scanning Electron Microscopy) and GUS staining analysis, ae1 mutation enhanced as1/as2 phenotype in vein differentiation and ectopic expression of KNOX gene, which suggest that AE1 is invovled in AS1-AS2 regulation network. AE1 gene has been cloned by map-based cloning, and the gene encodes an RNA-dependent RNA polymerase (RdRP), which sequence is identical to those of previously identified genes, SGS2 and SDE1. The expression of AE1 is ubiquitous. We found there was no direct regulatory relationship between AS2 and AE1 in transcriptional level. Our northern blotting experiment revealed that abnormalities in as2 ae1 were accompanied by high levels of microRNA165 in the as2 ae1 leaves, which led to mRNA cleavage of genes in the HD-ZIP III family. The products of cleavage are relatively stable in vivo. All these results indicate that AE1 and AS2/AS1 synergically regulate downstream gene,and AE1 participates in the AS2-AS1 network regulating leaf development, AE1 is invovled in microRNA regulation. Our results showed that AE1 and AS1/AS2 are partially redundant. Therefore, leaf adaxial identity is formed through a pathway of up-stream regulators AS1-AS2 and AE1, and down-stream target PHB linked by miR165. We proposed that RdRP is involved in regulation endogenous genes by synthesis dsRNA, the systemic silencing signal affect the expression of MIR165 and KNOX. AS1/AS2 as transcriptional factors may regulate downstream genes by specific-seqence recognition, or associates with components in an epigenetic control of gene expression.
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