The proteolytic function of the Arabidopsis 26S proteasome is required for specifying leaf adaxial identity.

Polarity formation is central to leaf morphogenesis, and several key genes that function in adaxial-abaxial polarity establishment have been identified and characterized extensively. We previously reported that Arabidopsis thaliana ASYMMERTIC LEAVES1 (AS1) and AS2 are important in promoting leaf adaxial fates. We obtained an as2 enhancer mutant, asymmetric leaves enhancer3 (ae3), which demonstrated pleiotropic plant phenotypes, including a defective adaxial identity in some leaves. The ae3 as2 double mutant displayed severely abaxialized leaves, which were accompanied by elevated levels of leaf abaxial promoting genes FILAMENTOUS FLOWER, YABBY3, KANADI1 (KAN1), and KAN2 and a reduced level of the adaxial promoting gene REVOLUTA. We identified AE3, which encodes a putative 26S proteasome subunit RPN8a. Furthermore, double mutant combinations of as2 with other 26S subunit mutations, including rpt2a, rpt4a, rpt5a, rpn1a, rpn9a, pad1, and pbe1, all displayed comparable phenotypes with those of ae3 as2, albeit with varying phenotypic severity. Since these mutated genes encode subunits that are located in different parts of the 26S proteasome, it is possible that the proteolytic function of the 26S holoenzyme is involved in leaf polarity formation. Together, our findings reveal that posttranslational regulation is essential in proper leaf patterning.