BACKGROUND : The NAC family of transcription factors is one of the largest gene families of transcription factors in
plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis. These transcription
factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress
induced processes has received relatively little attention. This study investigates NAC family transcription factors
involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato.
RESULTS : The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities,
several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426:
183–196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on
the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03
exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were
identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3’H and PaLAR3
were consistently down regulated in the overexpression lines. In accordance, the overexpression lines showed
reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by
directly interacting with the promoter of the repressed genes. However, transactivation studies of PaNAC03 and
PaLAR3 in Nicotiana benthamiana showed that PaNAC03 activated PaLAR3A, suggesting that PaNAC03 does not act
as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid
CONCLUSIONS : PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC
genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated
secondary metabolite production.
Additional file 1: Sequences of primers used in the study.
Additional file 2: Scripts used for Nesoni, tophat, cufflinks and cuffdiff.
Additional file 3: Figure S2. Schematic representation of the PaLAR3A
promoter (Genbank accession no. KX574229.1) in black and the PaLAR3B
promoter (KX574230.1) in red, the white regions in PaLAR3B promoter
corresponds to deletions in the sequence compared to PaLAR3A promoter.
The two NAC binding sites (TTTCGT) present in the region unique to the
PaLAR3A promoter are indicated in yellow. In the PaLAR3A_mut promoter it
is only these two sites which has been mutated in the remainder of the
PaLAR3A promoter is intact.
Additional file 4: Clustal W Alignment of Norway spruce subgroup III-3
NAC proteins. The coloured boxes correspond to the conserved N-terminal
motifs A (light blue), B (pale green), C (pale red, D (lilac) and E (pale gold).
The shaded residues indicate residues conserved in the C-terminal region.
Additional file 5: Amino acid identity and similarity in subgroup III-3 NAC
proteins. Percent amino acid identity (above the diagonal) and similarity
(below the diagonal) in the complete protein sequences (A) or the C-terminal
part of the proteins (B).
Additional file 6: Relative expression of putative PaNAC3 overexpression
lines. The relative expression was determined in relation to the
untransformed wild type line 95:61:21.
Additional file 7: Transcriptional regulation of PaNAC3 in response to
standard maturation treatment in the wild type line 95:61:21.
Additional file 8: Table S7. RNAseq metrics after Nesoni filtering.
Additional file 9: Alignment summaries from tophat.
Additional file 10: Enriched GO terms among consistently down- or
up-regulated genes in PaNAC3 overexpression lines.
Additional file 11: Consistently up-regulated genes in PaNAC3 overexpression
Additional file 12: Consistently down-regulated genes in PaNAC3 overexpression