Extended Data Fig. 7. Transient overexpression of GATA TFs promotes the expression of genes containing the GATC motif.

(a) Experimental setup: Arabidopsis leaf protoplasts were isolated and transformed by PEG-mediated delivery with plasmids carrying GFP, GATA1, GATA4, or GATA6 under the control of a double 35S promoter. RNA was extracted for RNA-Seq analysis eight hours after transformation. Two biological repeats of protoplast isolation were done, with each plasmid transformed twice per isolation replicate, resulting in four replicates per plasmid. In each set of protoplast isolation, an additional sample was transformed with the GFP plasmid to estimate transformation efficiency, which was determined by imaging to be 59% and 69.7% at eight hours post-transformation. (b) Verification of overexpression (OE): Gene expression levels of GATA1, GATA4, GATA6, and GFP (as indicated in the panel title) are shown. Expression levels are log2-transformed and plotted for the four plasmids across all four replicates. Note the low level of GFP detection in the GATA TF samples is due to leaky expression of a YFP selection gene driven by a seed coat promoter also found in the plasmids; due to sequence similarities between YFP and GFP, there is cross-alignment of sequencing reads. (c) Differential expression analysis: A volcano plot shows the -log10 adjusted Wald test p-values relative to the log2 fold changes in gene expression following OE of each GATA TF compared to GFP OE. Calculations were performed using DESeq2 based on four replicates per experiment⁶⁶. Genes showing at least a 50% change in expression with an adjusted p-value < 0.001 were defined to be differentially expressed and are shown in blue (downregulated) and red (upregulated); all other genes are shown in gray. (d) The percentage of genes with a GATC motif within 500 bp downstream of the TSS is shown. These are categorized based on increased, decreased, or unchanged expression (labeled ‘Unchanged’) as defined in c. Further separation is provided for genes with 1, 2, or 3 or more GATC motifs for each GATA TF OE experiment.