Opposite action of R2R3-MYBs from different subgroups on key genes of the shikimate and monolignol pathways in spruce

Abstract

Redundancy and competition between R2R3-MYB activators and repressors on common target genes has been proposed as a fine-tuning mechanism for the regulation of plant secondary metabolism. This hypothesis was tested in white spruce [Picea glauca (Moench) Voss] by investigating the effects of R2R3-MYBs from different subgroups on common targets from distinct metabolic pathways. Comparative analysis of transcript profiling data in spruces overexpressing R2R3-MYBs from loblolly pine (Pinus taeda L.), PtMYB1, PtMYB8, and PtMYB14, defined a set of common genes that display opposite regulation effects. The relationship between the closest MYB homologues and 33 putative target genes was explored by quantitative PCR expression profiling in wild-type P. glauca plants during the diurnal cycle. Significant Spearman's correlation estimates were consistent with the proposed opposite effect of different R2R3-MYBs on several putative target genes in a time-related and tissue-preferential manner. Expression of sequences coding for 4CL, DHS2, COMT1, SHM4, and a lipase thio/esterase positively correlated with that of PgMYB1 and PgMYB8, but negatively with that of PgMYB14 and PgMYB15. Complementary electrophoretic mobility shift assay (EMSA) and transactivation assay provided experimental evidence that these different R2R3-MYBs are able to bind similar AC cis-elements in the promoter region of Pg4CL and PgDHS2 genes but have opposite effects on their expression. Competitive binding EMSA experiments showed that PgMYB8 competes more strongly than PgMYB15 for the AC-I MYB binding site in the Pg4CL promoter. Together, the results bring a new perspective to the action of R2R3-MYB proteins in the regulation of distinct but interconnecting metabolism pathways.

Keywords: Conifers; R2R3-MYB evolution; phenylpropanoid pathway; protein–DNA binding; transcriptional network..

Fig. 1.

Comparative analysis of microarray profiles from Pinus taeda PtMYB1, PtMYB8, and PtMYB14 overexpression (OE) in spruce. (A) Numbers of shared and unique misregulated sequences in PtMYB1, PtMYB8, and PtMYB14OE. (B) Expression fold change (FC) of the 70 common misregulated sequences compared with the wild type. Red bars: opposite FC to MYB1-OE. See Table S2 for sequence IDs. (C) KEGG functional categories (%) of the 70 common sequences misexpressed in all three transgenic lines. For details, see Supplementary Table S1 at JXB online.

Fig. 2

Analysis of PgMYB8 and PgMYB15 binding to AC elements present in Pg4CL and PgDHS2 promoters in spruce. (A, B) Upstream flanking and 5′ UTR sequences (450bp) of the spruce (A) Pg4CL and (B) PgDHS2 genes. For both Pg4CL and PgDHS2 sequences, underlined nucleotides correspond to the 30bp probes (containing either an AC-I or AC-II element) designed for EMSA with PgMYB8, PgMYB14, and PgMYB15 recombinant proteins. The sequences shown represent the regions in which AC elements were identified in the 4CL promoter (1885bp) and DHS2 promoter (1353bp). Nucleotide representations are as follows: bold in upstream regions; italics in 5′ UTR sequences; AC-I sequences are boxed and AC-II sequences have a double box (ACCAACT) or a dashed box (ACCAACC). (C, D) EMSA testing of the binding of PgMYB8 (C) and PgMYB15 (D) recombinant protein to the labelled AC-I- and AC-II-containing probe [30bp fragments from Pg4CL (top) and PgDHS2 (bottom) promoters; see Fig. 3 for details]. Competition analyses used unlabelled probes containing AC-I, ACII, or mutated AC elements (mAC). Electrophoretic shifts (bound) and free DNA probes are indicated by arrows. (This figure is available in colour at JXB online.)

Fig. 3.

DNA binding competition analysis between PgMYB8 and PgMYB15 for an AC-I-containing promoter fragment from Pg4CL. EMSA using MYB8 and MYB15 recombinant proteins and labelled oligonucleotides from Pg4CL with a variable amount of (A) PgMYB8 protein or (B) PgMYB15 protein. Increasing stoichiometric ratios of MYB recombinant protein are indicated. Electrophoretic shifts (bound) and free DNA probes are indicated by arrows. Exposure showing the best resolution of the specific TF–DNA complex was used for each panel.

Fig. 4.

Proposed action of MYBs in the regulatory network controlling primary and secondary metabolism in conifers. An antagonistic action of PgMYB8 (activation) and PgMYB15 (repression) on interconnecting metabolic pathways is underlined (in bold): DHS2, 4CL, SHM4, and COMT are identified (boxed) as target genes under the control of both MYBs. Dashed lines indicate putative indirect effects of PgMYB15 on flavonoid and anthocyanin metabolic pathways. The network is also thought to involve PgMYB1 and PgMYB14, among others, which have not yet been tested in promoter binding assays but produce similar results as PgMYB8 (activation) and PgMYB15 (repression) in co-transformation assays, respectively. Genes that may fall under the antagonistic action of other MYBs are in parentheses. PHE, phenylalanine; TYR, tyrosine; TRP, tryptophan. (This figure is available in colour at JXB online.)