TCP4 directly activates <i>HAT2</i> transcription.

(A) Comparative transcript levels of Class II HD-ZIP genes in Arabidopsis seedlings in previously reported microarray datasets upon auxin treatment using the signature tool of Genevestigator database (https://www.genevestigator.com/gv/plant.jsp). Top 9 transcriptome profiles are shown. (B) and (D) Relative transcript levels of class II HD-ZIP genes in jaw-D;ProTCP4:mTCP4:GR (B) and HAT2 in indicated genotypes (D) determined by RT-qPCR in 9-day old seedlings grown without dexamethasone (Mock) or in the continuous presence 12 μM dexamethasone (DEX). (C) RT-qPCR analysis of HAT2 expression in 9-day old jaw-D;ProTCP4:mTCP4:GR seedlings treated with 12 μM dexamethasone for indicated durations. (E) RT-qPCR analysis of indicated transcripts in 9-day old jaw-D;ProTCP4:mTCP4:GR seedlings treated either with DEX alone or with DEX plus and combination of NPA and L-Kyn, pharmacological inhibitors of auxin transport and synthesis respectively, for 2 hrs. Solvent treatment (Mock) has been used a negative control. (F) HAT2 transcript levels determined by RT-qPCR in 9-day old seedlings grown without (Mock) or with (DEX) 12 μM dexamethasone. j;GR X C indicate jaw-D;ProTCP4:mTCP4:GR X Col-0 and and j;GR X s indicate jaw-D;ProTCP4:mTCP4:GR X shy2-2. Transcript levels were initially normalized to PP2A/TUB2 internal control and then compared to respective mock controls. (G) and (H) HAT2 transcript levels analyzed by RT-qPCR in 9-day old jaw-D;GR (jaw-D;ProTCP4:mTCP4:GR) (G) or Pro35S:GR (Pro35S:mTCP4:GR) (H) seedlings treated either with 40 μM cyclohexamide (CHX) or with a combination of 40 μM cyclohexamide and 20 μM dexamethasone (CHX DEX) for 4 hrs. (I) Schematic representation of the HAT2 locus. Exons are shown in gray box and translation start site is indicated with an arrow. BS1-BS3 represent the putative TCP4-binding motifs that are used for EMSA experiments in (J). R1-R4 indicate the regions that are used for ChIP-qPCR analysis shown in (K). (J) Electrophoretic mobility shift assay demonstrating the retardation of the oligonucleotides corresponding to the TCP4-binding sites in the HAT2 locus shown in (I) by recombinant MBP-fused TCP4 domain (MBP-TCP4D). 250-fold of un-labeled oligonucleotides was used as competitors. For super shift, anti-MBP antibody was used. (K) ChIP-qPCR analysis of the HAT2 chromatin regions R1-R4 (shown in I) with anti-FLAG antibody. LOX2 and TUB2 were used as positive and negative controls, respectively. For RT-qPCR analyses in (B-H) and Chip-qPCR in (K), averages of biological triplicates are shown. Error bars indicate SD. * indicates p t-test was used.