Characterization of Evolutionarily ConservedMotifs Involved in Activity and Regulation of theABA-INSENSITIVE （ABI） 4 Transcription Factor

In recent years, the transcription factor ABI4 has emerged as an important node of integration for externaland internal signals such as nutrient status and hormone signaling that modulates critical transitions during the growthand development of plants. For this reason, understanding the mechanism of action and regulation of this protein rep-resents an important step towards the elucidation of crosstalk mechanisms in plants. However, this understanding hasbeen hindered due to the negligible levels of this protein as a result of multiple posttranscriptional regulations. To betterunderstand the function and regulation of the ABI4 protein in this work, we performed a functional analysis of severalevolutionarily conserved motifs. Based on these conserved motifs, we identified ortholog genes of ABI4 in differentplant species. The functionality of the putative ortholog from Theobroma cacao was demonstrated in transient expres-sion assays and in complementation studies in plants. The function of the highly conserved motifs was analyzed aftertheir deletion or mutagenesis in the Arabidopsis ABI4 sequence using mesophyll protoplasts. This approach permitted usto immunologically detect the ABI4 protein and identify some of the mechanisms involved in its regulation. We identi-fied sequences required for the nuclear localization （AP2-associated motif） as well as those for transcriptional activationfunction （LRP motif）. Moreover, this approach showed that the protein stability of this transcription factor is controlledthrough protein degradation and subcellular localization and involves the AP2-associated and the PEST motifs. We dem-onstrated that the degradation of ABI4 protein through the PEST motif is mediated by the 26S proteasome in responseto changes in the sugar levels.