CEPD proteins activate N starvation response genes by interfering with the repressive activity of transcription factors TGA1 and TGA4

Plants have developed a variety of adaptive responses to variable nitrogen (N) conditions in the soil. N starvation leads to profound alterations in the transcriptome, allowing improved N acquisition, allocation and remobilization. In Arabidopsis thaliana, N starvation-induced CEPD (C-TERMINALLY ENCODED PEPTIDE DOWNSTREAM) proteins have been shown to be required for transcriptional activation of genes involved in high affinity nitrate transport. CEPDs belong to a plant-specific class of glutaredoxin-like proteins (called ROXYs in A. thaliana) that interact with TGACG-binding transcription factors (TGAs). Here show that TGA1 and TGA4 act as molecular links between CEPDs (ROXYs 6-9) and target promoters. CEPDs permit activation of gene expression by binding to TGA1/4 at target promoters, thereby interfering with a strong repression mechanism that is recruited by TGA1/4. Although CEPDs resemble glutaredoxins with glutathione-dependent oxidoreductase activity, a ROXY9 variant with a cysteine to serine exchange at the putative catalytic cysteine can confer low nitrogen-induced target gene expression. This finding excludes that ROXY9 functions by altering the redox state of target proteins. Still, changing the conserved binding site from CCLC to CPYC abrogated ROXY9 activity suggesting that the PY sequence may interfere with ROXY9-protein interactions that are required to render the TGA1/4-mediated repressive mechanism dysfunctional. Overall design: RNA was prepared from roots of Col-0 wild-type and roxy6,7,8,9 quadruple mutant (cepd) plants subjected to LN conditions. Samples from four independent experiments with each sample containing roots from 50 plantlets grown on five separate plates were analyzed. For Col-0, we had to exclude one data set since the corresponding sample had been contaminated with leaf material.