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Consistently up-regulated genes in PaNAC3 overexpression lines. (DOCX 15Â kb)

Relative expression of putative PaNAC3 overexpression lines. The relative expression was determined in relation to the untransformed wild type line 95:61:21. (DOCX 53.1Â kb)

RNAseq metrics after Nesoni filtering. (DOCX 19Â kb)

Alignment summaries from tophat. (DOCX 11Â kb)

Transcriptional regulation of PaNAC3 in response to standard maturation treatment in the wild type line 95:61:21. (PDF 447Â kb)

Transcription factors (TFs) forming MYB-bHLH-WDR complexes are known to regulate the biosynthesis of specialized metabolites in angiosperms through an intricate network. These specialized metabolites participate in a wide range of biological processes including plant growth, development, reproduction as well as in plant immunity. Studying the regulation of their biosynthesis is thus essential. While MYB (TFs) have been previously shown to control specialized metabolism (SM) in gymnosperms, the identity of their partners, in particular bHLH or WDR members, has not yet been revealed. To gain knowledge about MYB-bHLH-WDR transcription factor complexes in gymnosperms and their regulation of SW, we identified two bHLH homologs of AtTT8, six homologs of the MYB transcription factor AtTT2 and one WDR ortholog of AtTTG1 in Norway spruce. We investigated the expression levels of these genes in diverse tissues and upon treatments with various stimuli including methyl-salicylate, methyl-jasmonate, wounding or fungal inoculation. In addition, we also identified protein-protein interactions among different homologs of MYB, bHLH and WDR. Finally, we generated transgenic spruce cell lines overexpressing four of the Norway spruce AtTT2 homologs and observed differential regulation of genes in the flavonoid pathway and flavonoid contents.

Norway spruce [Picea abies (L.) Karst.] is a main tree species in European forests and is important both ecologically and economically. The root rot fungus Heterobasidion annosum sensu lato (s.l.) is the main P. abies pathogen. Including resistance in breeding programs would help mitigating the impact of the pathogen but knowledge regarding defense mechanisms in P. abies needs a better understanding. The work within this thesis intended to expand the existing knowledge on P. abies resistance mechanisms focusing on hormone signaling, flavonoid biosynthesis and its transcriptional regulation. I found that jasmonic acid is the major hormone controlling defense signaling pathways in P. abies. Furthermore, we validated a candidate gene, PaLAR3, as a resistance marker for H. annosum s.l. in P. abies. PaLAR3 encodes an enzyme responsible for the synthesis of (+) catechin, which showed a fungistatic effect on H. parviporum. Analysis of genetic diversity revealed two allelic lineages of PaLAR3 that showed significant differences in fungal resistance and (+) catechin content that were explained by dissimilarities in inducibility. We studied the role of PaNAC03, a transcription factor that is associated with H. annosum s.l. infection. PaNAC03 not only showed repression of multiple genes including PaLAR3, but bound only to the promoter of one of the PaLAR3 allelic lineages explaining at least partly the differences in allelic expression that were observed. Finally, we identified a full repertoire of members of a MYB/bHLH/WDR transcription factor complex in Norway spruce, which showed differences in protein interactions and expression patterns, and also in ability to control the expression of genes in the flavonoid biosynthetic pathway including PaLAR3.

Background Heterotrimeric G-proteins are important signalling switches, present in all eukaryotic kingdoms. In plants they regulate several developmental functions and play an important role in plant-microbe interactions. The current knowledge on plant G-proteins is mostly based on model angiosperms and little is known about the G-protein repertoire and function in other lineages. In this study we investigate the heterotrimeric G-protein subunit repertoire in Pinaceae, including phylogenetic relationships, radiation and sequence diversity levels in relation to other plant linages. We also investigate functional diversification of the G-protein complex in Picea abies by analysing transcriptional regulation of the G-protein subunits in different tissues and in response to pathogen infection. Results A full repertoire of G-protein subunits in several conifer species were identified in silico. The full-length P. abies coding regions of one Gα-, one Gβ- and four Gγ-subunits were cloned and sequenced. The phylogenetic analysis of the Gγ-subunits showed that PaGG1 clustered with A-type-like subunits, PaGG3 and PaGG4 clustered with C-type-like subunits, while PaGG2 and its orthologs represented a novel conifer-specific putative Gγ-subunit type. Gene expression analyses by quantitative PCR of P. abies G-protein subunits showed specific up-regulation of the Gα-subunit gene PaGPA1 and the Gγ-subunit gene PaGG1 in response to Heterobasidion annosum sensu lato infection. Conclusions Conifers possess a full repertoire of G-protein subunits. The differential regulation of PaGPA1 and PaGG1 indicates that the heterotrimeric G-protein complex represents a critical linchpin in Heterobasidion annosum s.l. perception and downstream signaling in P. abies. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0676-1) contains supplementary material, which is available to authorized users.

Primer sequences for full-length amplification of the Gα-, Gβ- and Gγ-subunits cloning of the sequences and qPCR. (DOCX 17 kb)

Predictions of alpha-helices in short Gγ-subunits PaGG1, PaGG2, GG1 and GG2. The GGL domain (pfam00631) is underlined. The PreSSAPro software ( http://bioinformatica.isa.cnr.it/PRESSAPRO/ ) was used to predict the formation of α-helices based on amino acid propensities, residues likely to form α-helices are shown in bold font and are boxed, residues shared between three or more proteins are shaded and asterisks indicate conserved amino acids. (PDF 10 kb)

Accession numbers for the Gα-, Gβ- and Gγ-subunits sequences found in the plant kingdom. Sequences have been either cloned or retrieved from the Gene Index Project (The Gene Index Databases-Dana Faber Cancer Institute; Lee et al., [54]; Pertea et al., [55]; Quackenbush et al., [56] ; Tsai et al., [64]), NCBI EST/Nucleotide/Protein, Phytozome v9.1 (Goodstein et al., [57]) or Uniprot [65]; underlined sequences were used for primer design. (DOCX 18 kb)

Phylogeny of Gα- and Gβ-subunits of the plant kingdom. The figure shows neighbor-joining trees of full-length sequence alignments of the Gα- (a) and Gβ-subunits. (b) including sequences from species of the Brassicaceae (blue), Fabaceae (orange) and Pinaceae (purple) and the moss Physcomitrella patens (green, root). Bootstrap support over 65 is indicated at the nodes. (PDF 502 kb)

Alignment of Gy-subunit sequences. (PDF 8038 kb)

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 biosynthetic genes. 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. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0952-8) contains supplementary material, which is available to authorized users.