Figure 3.

Two NAC TFs Bind to a Conserved Promoter Sequence to Activate EXPA2 Transcription.

(A) Construct used for functional analysis of the EXPA2 conserved sequence (EXPA2-CS; black box) and the control construct containing only the minimal promoter (Min; gray box).

(B) Seeds from two representative transgenic lines for each construct (Min or 4xEXPA2-CS) were imbibed for 24 h. Average luciferase levels and SE for at least 10 seeds for each line are shown.

(C) Sequence of the wild-type EXPA2-CS and a version with mutations in the putative NAC binding sites (EXPA2-CSm). Mutated bases are indicated in lowercase.

(D) Yeast strains containing one copy of the EXPA2-CS or EXPA2-CSm fused to the HIS3 reporter gene were mated to strains containing the AD-∅, AD-GFP, AD-NAC1L, or AD-NAC25 constructs. Diploid cells were grown on diploid (-L-W) and screening (-L-W-H) plates with increasing concentrations of 3-AT.

(E) Diagram of the EXPA2 promoter (reporters) and TF (effectors) constructs used for transient expression analyses in N. benthamiana leaves. N. benthamiana leaves were agroinfiltrated with the ProEXPA2 or the ProEXPA2m construct together with an effector construct overexpressing NAC25 or NAC1L.

(F) Control is the empty effector plasmid (pCX) co-expressed with each of the reporters. Average luciferase activities and SE from at least six replicates are shown, and similar results were obtained in an additional agroinfiltration experiment. Asterisks represent significant differences at **P < 0.01 and *P < 0.05 using the Mann–Whitney test (two-sided).