Fig. 4: Transcription factor NtMYB28 regulates hydroxycinnamic acid biosynthesis and the expression of genes involved in phenylpropanoid pathway.

a The sub-network extracted from the GMRN for chlorogenic acid biosynthesis. SG, structural genes. TF, transcription factors. ME, metabolites. b Expression patterns of MEs, SGs, and TFs. “4” and “-4” represent the maximum and minimum value of the Z-score standardized expression, respectively. DAT, days after topping. c Transcriptional activation assay of NtMYB28 in yeast. d Transient expression assay showing transcriptional activation of the LUC reporter gene (driven by the promoter of Nt4CL2A and NtPAL2) by NtMYB28 (n = 5 biological replicates). e Specific binding of NtMYB28 protein to promoters of Nt4CL2A and NtPAL2 by one-hybrid system using bait and either prey or negative control. f EMSA with NtMYB28 protein performed with the probes derived from the Nt4CL2A and NtPAL2 promoter. g, h RNA-seq and RT-qPCR showing that Nt4CL2A (g) and NtPAL2 (h) are positively regulated by NtMYB28 (n = 3 biological replicates). i Comparison of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid and caffeic acid mass spectrometry data of WT, NtMYB28-OE and ntmyb28-KO tobaccos. j Chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid and caffeic acid contents in leaves of WT, NtMYB28-OE and ntmyb28-KO tobaccos (n = 3 biological replicates). k GMRN accurately predicts targets of NtMYB28, as demonstrated by the identification of differentially expressed genes (DEGs) in the TF mutant and the overexpression lines. l Heatmap analysis of the genes related to phenylpropanoid pathway. “2” and “-2” represent the maximum and minimum value of the Z-score standardized expression, respectively. m KEGG pathway enriched by DEGs between WT, NtMYB28-OE and ntmyb28-KO tobaccos. In (c), (e), and (f), the images were representative of three independent repeats with similar results. In (d), (g), (h), and (j), error bars represent the means ± standard errors of biological replicate. A two-tailed Student’s t test was used to determine P values. Different lowercase letters indicate significant differences (P < 0.001). In (k) and (m), the P values were calculated using hypergeometric distribution (two-sided) and then adjusted using the FDR. Source data are provided as a Source Data file.