Characterization of Evolutionarily Conserved Motifs Involved in Activity and Regulation of the ABA-INSENSITIVE (ABI) 4 Transcription Factor

In recent years, the transcription factor ABI4 has emerged as an important node of integration for external and internal signals such as nutrient status and hormone signaling that modulates critical transitions during the growth and development of plants. For this reason, understanding the mechanism of action and regulation of this protein represents an important step towards the elucidation of crosstalk mechanisms in plants. However, this understanding has been hindered due to the negligible levels of this protein as a result of multiple posttranscriptional regulations. To better understand the function and regulation of the ABI4 protein in this work, we performed a functional analysis of several evolutionarily conserved motifs. Based on these conserved motifs, we identified ortholog genes of ABI4 in different plant species. The functionality of the putative ortholog from Theobroma cacao was demonstrated in transient expression assays and in complementation studies in plants. The function of the highly conserved motifs was analyzed after their deletion or mutagenesis in the Arabidopsis ABI4 sequence using mesophyll protoplasts. This approach permitted us to immunologically detect the ABI4 protein and identify some of the mechanisms involved in its regulation. We identified sequences required for the nuclear localization (AP2-associated motif) as well as those for transcriptional activation function (LRP motif). Moreover, this approach showed that the protein stability of this transcription factor is controlled through protein degradation and subcellular localization and involves the AP2-associated and the PEST motifs. We demonstrated that the degradation of ABI4 protein through the PEST motif is mediated by the 26S proteasome in response to changes in the sugar levels.