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. 2021 Feb 10;12:642758. doi: 10.3389/fpls.2021.642758

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Copyright © 2021 Cabral, Masuda, Ballesteros, de Almeida-Engler, Alves-Ferreira, De Toni, Bizotto, Ferreira and Hemerly.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Characterization of the interaction between ABAP1 and TCP16 and their role in transcription. (A) Yeast two hybrid interaction assays between ABAP1 and cloned in both AD and BD vectors. Negative controls used DAD and DBD empty vectors. The panel shows conjugated yeasts growing in SD medium lacking leucine, tryptophan, histidine and adenine, for selection of strong protein interactions. (B) GST-pull down assay between ABAP1–GST and TCP16-HIS produced in E. coli. Western blot analysis used anti-HIS antibody. Lane 1, TCP16-HIS with ABAP1-GST; lane 2, TCP16-HIS with GST alone; lanes 3 and 4, 1/10 inputs of ABAP1-GST and TCP16-HIS, respectively. (C) EMSA of ABAP1, TCP16 and the complex TCP16–ABAP1 with wild-type (WT) and mutated (mut) radiolabeled probes of CDT1b promoter regions harboring TCP recognition box. Lane 1, TCP16 binds to CDT1b WT probe; lane 2, addition of ABAP1 causes a supershift in the TCP16 binding to CDT1b WT probe; GST alone (lane 3) or ABAP1 alone (lane 4) do not bind to CDT1b WT probe; lane 5, TCP16 does not bind to CDT1b mutated probe. (D,E) Chromatin immunoprecipitation of wild-type Col (WT) and ABAP1^OE flower buds with anti-ABAP1. (D) Schematic representation of the amplified regions of CDT1b. Black triangle indicates the class I TCP motif, located at position –130 to –136 bp. Red arrows and numbers indicate primers used for ChIP-qPCR assays, and short green line indicates DNA probe used for EMSA. The translational start site (ATG) is shown at position +1. (E) Chromatin immunoprecipitated with anti-ABAP1 antibody was analyzed by qPCR using gene-specific primer sets, numbered as indicated in (D). The graph shows the fold enrichment of qPCR amplified regions at the promoter (1, 2), and 3’UTR (3,4) of CDT1b. The fold enrichment of each amplified region was calculated as a ratio between IP ABAP1^OE and IP WT of values normalized with values of the ACTIN 2 promoter. Error bars represent the SD of three biological replicates. Asterisk (^∗) indicates significant difference from the enrichment according to Student’s t test (p-value < 0.05). IP, immunoprecipitated with anti-ABAP1 antibody.