of 19 bp, namely, the secondary wall NAC-binding element (SNBE) with (T/A)NN
(C/T)(T/C/G)TNNNNNNNA(A/C)GN(A/C/T)(A/T) as a consensus sequence. This ciselement is present in many promoter genes involved in secondary wall biosynthesis, cell
wall modification and programmed cell death (Zhong et al. 2010b; Wang et al. 2011),
and the element is conserved in different species, including poplar, rice, eucalyptus and
maritime pine (Ohtani et al. 2011; Zhong et al. 2011b; Pascual et al. 2017). It has also
been shown that the DNA sequences flanking the core motif are in contact with the
NAC domain (Welner et al. 2012) and contribute to both the affinity and the specificity
of the target promoter binding.
For NAC proteins, in addition to regulating the expression of several target genes
involved in multiple cellular processes, their expression can be regulated at three
levels:
a. Transcriptional regulation at the specific TF binding level. The promoter region
of PpNAC2 and PpNAC3, two stress-responsive NAC genes of Pinus pinaster,
contain several cis-elements involved in stress response: DPBF1 (ABAresponsive element); MYB binding sites; dehydration and low-temperature
responsive elements, hormonal-responsive elements (jasmonic acid and salicylic
acid) and ethylene-responsive GCC-boxes; and binding site of ERF1 transcription
factors. Additionally, W-boxes were identified as being present in the promoter of
defence genes (Rushton et al. 1996; Eulgem et al. 1999; Maleck et al. 2000).
These cis-elements are the recognition sites for WRKY transcription factors
(Eulgem et al. 1999).
b. miRNA-mediated cleavage or alternative splicing at the post-transcriptional level.
NAC proteins are also potential targets of miRNAs. It has been observed that the
miR164-targeted NAC domain TF module is conserved across species. For example, CUC1 and CUC2 of Arabidopsis are post-transcriptionally downregulated
by miR164 cleavage, while PaNAC01 of P. abies, a putative orthologue of CUC
NAC genes of Arabidopsis, also harbours the miR164 recognition site. However,
no miR164 and no NAC protein within the binding site for this microRNA has
been identified in primitive vascular plants, such as S. moellendorffii and P. patens
(Axtell et al. 2007). This finding suggests that this type of post-transcriptional regulation appeared before the separation between gymnosperm and angiosperms but
after the origin of seed plants.
In contrast, in Populus, it has been shown that PtrSND1, a gene involved in
secondary cell wall biosynthesis, has alternative splicing variants that lack DNAbinding and transactivation abilities but retain dimerization capability. This splice
variant protein (PtrSND1-A2) is a dominant negative of PtrSND1 and can antagonize the autoregulation of its PtrSND1 member genes, thus contributing to transcriptional homeostasis associated with wood formation and plant growth (Li et al. 2012).
c. NAC post-translational modifications. The NAC proteins can also undergo
post-translational modifications such as ubiquitination, phosphorylation, proteolysis, dimerization and/or interaction with other proteins (Xie et al. 2002;
Puranik et al. 2012). NTL6 is a membrane-associated NAC TF that has been
implicated in cold-induced pathogen resistance, and its activity is regulated with
NAC Transcription Factors in Woody Plants
205
(C/T)(T/C/G)TNNNNNNNA(A/C)GN(A/C/T)(A/T) as a consensus sequence. This ciselement is present in many promoter genes involved in secondary wall biosynthesis, cell
wall modification and programmed cell death (Zhong et al. 2010b; Wang et al. 2011),
and the element is conserved in different species, including poplar, rice, eucalyptus and
maritime pine (Ohtani et al. 2011; Zhong et al. 2011b; Pascual et al. 2017). It has also
been shown that the DNA sequences flanking the core motif are in contact with the
NAC domain (Welner et al. 2012) and contribute to both the affinity and the specificity
of the target promoter binding.
For NAC proteins, in addition to regulating the expression of several target genes
involved in multiple cellular processes, their expression can be regulated at three
levels:
a. Transcriptional regulation at the specific TF binding level. The promoter region
of PpNAC2 and PpNAC3, two stress-responsive NAC genes of Pinus pinaster,
contain several cis-elements involved in stress response: DPBF1 (ABAresponsive element); MYB binding sites; dehydration and low-temperature
responsive elements, hormonal-responsive elements (jasmonic acid and salicylic
acid) and ethylene-responsive GCC-boxes; and binding site of ERF1 transcription
factors. Additionally, W-boxes were identified as being present in the promoter of
defence genes (Rushton et al. 1996; Eulgem et al. 1999; Maleck et al. 2000).
These cis-elements are the recognition sites for WRKY transcription factors
(Eulgem et al. 1999).
b. miRNA-mediated cleavage or alternative splicing at the post-transcriptional level.
NAC proteins are also potential targets of miRNAs. It has been observed that the
miR164-targeted NAC domain TF module is conserved across species. For example, CUC1 and CUC2 of Arabidopsis are post-transcriptionally downregulated
by miR164 cleavage, while PaNAC01 of P. abies, a putative orthologue of CUC
NAC genes of Arabidopsis, also harbours the miR164 recognition site. However,
no miR164 and no NAC protein within the binding site for this microRNA has
been identified in primitive vascular plants, such as S. moellendorffii and P. patens
(Axtell et al. 2007). This finding suggests that this type of post-transcriptional regulation appeared before the separation between gymnosperm and angiosperms but
after the origin of seed plants.
In contrast, in Populus, it has been shown that PtrSND1, a gene involved in
secondary cell wall biosynthesis, has alternative splicing variants that lack DNAbinding and transactivation abilities but retain dimerization capability. This splice
variant protein (PtrSND1-A2) is a dominant negative of PtrSND1 and can antagonize the autoregulation of its PtrSND1 member genes, thus contributing to transcriptional homeostasis associated with wood formation and plant growth (Li et al. 2012).
c. NAC post-translational modifications. The NAC proteins can also undergo
post-translational modifications such as ubiquitination, phosphorylation, proteolysis, dimerization and/or interaction with other proteins (Xie et al. 2002;
Puranik et al. 2012). NTL6 is a membrane-associated NAC TF that has been
implicated in cold-induced pathogen resistance, and its activity is regulated with
NAC Transcription Factors in Woody Plants
205