Insights into the transcriptional cascade involved in the initial and early phases of glandular trichome development in Nicotiana tabacum [DAPseq_MIXTA_ZFP8]

Glandular trichomes play a pivotal role in plant defense against a wide range of biotic and abiotic stresses. This study aims to employ AmMIXTA as a transcriptional switch to explore the genetic network that triggers glandular trichome development by leveraging its known role in promoting glandular trichome development when heterologously expressed in Nicotiana tabacum. To this end, we conducted a comprehensive RNA-seq analysis on transgenic tobacco lines expressing AmMIXTA under the control of a ß-estradiol-inducible promoter across key time points. Our findings reveal a significant upregulation of a suite of genes, predominantly transcription factors, following the induction of AmMIXTA, providing insights into the transcriptional cascade involved in the initial and early phases of glandular trichome development. Moreover, through confocal live cell imaging of transcriptional reporter lines, interaction studies and DAP-seq assays, we confirmed the involvement of NtZFP8, a gene identified in our study, in glandular trichome development. Our data support a synergistic model where AmMIXTA and NtZFP8 co-regulate a network of genes critical for glandular trichome formation. Our study opens new lead to investigate the complex genetic network controlling glandular trichome formation in tobacco and related Solanaceae species of agronomical interest. Overall design: In order to unravel downstream pathways regulated by AmMIXTA and NtZFP8 and so identify the transcriptional targets of both transcription factors, we employed DNA affinity purification sequencing (DAP-seq), a technique that facilitates the identification of DNA sequences directly bound by transcription factors (Bartlett et al., 2017). DNA reads from the NGS platform were filtered and mapped to the N.tabacum genome (Sierro et al., 2014) with an average mapping of 98% suggesting a high quality of the samples (Supplemental Table S4). Hits were identified by comparing against the negative control (nlsGFP protein), using criteria of a peak shape score greater than 2 and a p-value less than 0.05, to delineate significant binding regions.