Probing protein structural requirements for activation of membrane-bound NAC transcription factors in Arabidopsis and rice

Many transcription factors are stored in a dormant state through association with the cellular membranes in plants. Upon stimulation by internal and environmental signals, they are activated through proteolytic cleavage events either by membrane-associated proteases or by ubiquitination-dependent proteasome activities. Controlled proteolytic activation of the dormant, membrane-bound transcription factors (MTFs) is an intriguing way of ensuring rapid transcriptional responses to abrupt environmental changes. However, the underlying activation mechanisms and protein structural requirements are largely unknown in most cases. Here, we analyzed the primary and secondary structures of the NAC MTFs, particularly of the amino acid sequences surrounding the putative cleavage sites. Interestingly, the putative biologically active forms have strong hydrophilic motifs at their C-termini. Accordingly, transgenic Arabidopsis plants overexpressing the truncated forms having the C-terminal hydrophilic motifs exhibited distinct phenotypes. The finding was also applicable to rice NAC MTFs. Among the full-size OsNTL2 protein and a series of truncated OsNTL2 forms lacking the C-terminal transmembrane motif, only the OsNTL2 form (Os2ΔC4) having a strong hydrophilic peak at the C-terminus exhibited a high transcriptional activation activity when assayed in yeast cells. Our findings will provide insights into how plant MTFs are activated to release the biologically active forms.