RNAi suppression of RPN12a decreases the expression of type-A ARRs, negative regulators of cytokinin signaling pathway, in Arabidopsis

The 26S proteasome is a 2-MDa complex with a central role in protein turn over. The 26S proteasome is comprised of one 20S core particle and two 19S regulatory particles (RPs). The RPN12a protein, a non-ATPase subunit of the 19S RP, was previously shown to be involved in cytokinin signaling in Arabidopsis. To further investigate cellular roles of RPN12a, RNAi transgenic plants of RPN12a were constructed. As expected, the 35S:RNAi-RPN12a plants showed cytokinin signaling defective phenotypes, including abnormal formation of leaves and inflorescences. Furthermore, RNAi knock-down transgenic plants exhibited additional unique phenotypes, including concave and heart-shape cotyledons, triple cotyledons, irregular and clustered guard cells, and defects in phyllotaxy, all of which are typical for defective cytokinin signaling. We next examined the mRNA level of cytokinin signaling components, including type-A ARRs, type-B ARRs, and CRFs. The expression of type-A ARRs, encoding negative regulators of cytokinin signaling, was markedly reduced in 35S:RNAi-RPN12a transgenic plants relative to that in wild type plants, while type-B ARRs and CRFs were unaffected. Our results also indicate that in vivo stability of the ARR5 protein, a negative regulator of cytokinin signaling, is mediated by the 26S proteasome complex. These results suggest that RPN12a participates in feedback inhibitory mechanism of cytokinin signaling through modulation of the abundance of ARR5 protein in Arabidopsis.