Subgroup 4 R2R3-MYBs in conifer trees: gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses

Abstract

Transcription factors play a fundamental role in plants by orchestrating temporal and spatial gene expression in response to environmental stimuli. Several R2R3-MYB genes of the Arabidopsis subgroup 4 (Sg4) share a C-terminal EAR motif signature recently linked to stress response in angiosperm plants. It is reported here that nearly all Sg4 MYB genes in the conifer trees Picea glauca (white spruce) and Pinus taeda (loblolly pine) form a monophyletic clade (Sg4C) that expanded following the split of gymnosperm and angiosperm lineages. Deeper sequencing in P. glauca identified 10 distinct Sg4C sequences, indicating over-representation of Sg4 sequences compared with angiosperms such as Arabidopsis, Oryza, Vitis, and Populus. The Sg4C MYBs share the EAR motif core. Many of them had stress-responsive transcript profiles after wounding, jasmonic acid (JA) treatment, or exposure to cold in P. glauca and P. taeda, with MYB14 transcripts accumulating most strongly and rapidly. Functional characterization was initiated by expressing the P. taeda MYB14 (PtMYB14) gene in transgenic P. glauca plantlets with a tissue-preferential promoter (cinnamyl alcohol dehydrogenase) and a ubiquitous gene promoter (ubiquitin). Histological, metabolite, and transcript (microarray and targeted quantitative real-time PCR) analyses of PtMYB14 transgenics, coupled with mechanical wounding and JA application experiments on wild-type plantlets, allowed identification of PtMYB14 as a putative regulator of an isoprenoid-oriented response that leads to the accumulation of sesquiterpene in conifers. Data further suggested that PtMYB14 may contribute to a broad defence response implicating flavonoids. This study also addresses the potential involvement of closely related Sg4C sequences in stress responses and plant evolution.

Fig. 1.

Sequence analyses of angiosperm and conifer Sg4 R2R3-MYBs define the conifer specific subclade Sg4C (grey boxes) and identify associated amino acid motifs. (A and B) Rooted Neighbor–Joining trees were obtained with MEGA 4 software (Tamura et al., 2007) and Clustal W alignments of (A) the amino acid sequence (WRSLPKAAG in R2 to the predicted stop codon) or (B) the MYB DBD nucleotides (see Materials and methods). Bootstrap values >50% are shown. Other alignment methods and tree construction algorithms gave consistent results and are detailed in Supplementary Table S1 at JXB online. *Picea glauca (filled lozenges) and Pinus taeda (open lozenges) sequences reported here; known repressors of phenylpropanoid/lignin pathways are in bold. AtMYB3R4 and PttMYB3R are the outgroup, and AtMYB13, 20, and 123 (not of Sg4) are landmarks. Bars indicate the evolutionary distance as a percentage. (C) Amino acid motifs in the Sg4C C-termini ([1]–[6]) (MEME software). Top: MEME motif logos; bit scores indicate the information content for each position. Bottom: Clustal W alignment of predicted amino acid sequences and conserved motifs (shaded): G1 (EIPAFQ) and G2 (DF^F/_L^Q/_Gxx) are conserved in conifers; the C1 LIsrGIDPx^T/_SHRx^I/_L (Kranz et al., 1998) and the C2 motifs pdLNL^D/_ELxi^G/_S (Kranz et al., 1998, Jin et al., 2000) are conserved in all taxa (see A). Numbers in brackets are the amino acids positions (predicted from the MYB14 full-length sequence). (a) Angiosperms Sg4 sequences. (b) Other subgroup conifer sequences. (This figure is available in colour at JXB online.)

Fig. 2.

Stress-responsive transcript profiles of Sg4C sequences in P. taeda and P. glauca determined by RT-qPCR. (A–C) Transcript accumulation (fold change relative to controls) in 4-week-old plantlets in response to (A) mechanical wounding (after 0, 1.5, 6, and 24 h), (B) jasmonic acid (after 24 h) and (C) exposure to cold (24 h at 4 °C); AOC, allene oxide cyclase. (D) Transcript accumulation of PtMYB14, PgMYB14, and PgMYB15 in 2-year-old trees. N, needle; AS, apical stem (young elongating shoot); S2X, stem differentiating secondary xylem; WX: whole stem xylem; B, bark; RT, root tip. Data were from three biological replicates and were normalized relative to transcript levels of EF1-α (A–C) or both EF1-α and cdc2 (D). Significant Student's test at 0.05 (*), 0.01 (**), or 0.001 (***). (This figure is available in colour at JXB online.)

Fig. 3.

Promoter analysis and transgenic phenotypes with constitutive and tissue-preferential overexpression of PtMYB14. (A and B) Analysis of UBIproGUS and CADproGUS in 10 μm cross-sections from paraffin-embedded hypocotyls. (A) GUS staining (white arrow) in parenchyma, differentiating xylem, and ray cells with the UBIproGUS construct and (B) in differentiating xylem and ray cells with the CADproGUS construct. (C) Enzymatic MUG assays: means (±SE) for three biological replicates from the wild type (wt) and independent transgenic lines. (D–F) Morphology of 6-week-old in vitro plantlets. (D) Wild-type; (E) UBIpro PtMYB14-OE with hypertrophic hypocotyl and cotyledons with red pigmentation (black arrows in inset); (F) CADproPtMYB14-OE. (G–L) Cross-sections of 5 μm in paraffin-embedded hypocotyls of wild-type (G, J), UBIproPtMYB14-OE (H, K), and CADproPtMYB14-OE (I, L) hypocotyls showing polyphenolic parenchyma cells (white arrowhead) in both transgenics within the vasculature (H, I) and in outer subepidermal layers (K, L). Disorganized vasculature with large tracheids (*); starch grain accumulation in inner parenchyma cells (black arrows) in UBIproPtMYB14 transgenics (H). Bars: 50 μm (A, B), 5 mm (D–F), 100 μm (G–L). pm, parenchyma; ph, phloem; mx, metaxylem; pi, pith; rc, ray cell; ep, epidermis.

Fig. 4.

Transcript accumulation of the PtMYB14 transgene and other Sg4C sequences in hypocotyls and roots of 4-week-old wild-type and transgenic plantlets. Mean (±SE) of RT-qPCR determinations in control (wt), UBIproPtMYB14-OE (lines 16 and 18), and CADproPtMYB14-OE (lines 10 and 14) plantlets of four biological replicates, normalized relative to EF1-α transcripts. na, not amplified. Significant Student's test at 0.05 (*), 0.01 (**), or 0.001 (***).

Fig. 5.

Mono- and sesquiterpenoids in wild-type and PtMYB14-OE transgenic spruce (raw data). Monoterpenes (A, C, E) and (B, D, F) sesquiterpenes in wild-type (A, B), CADproPtMYB14 (C, D), and UBIproPtMYB14 (E, F) plantlets in three lines per transgenic and three replicates per line. Each value is a mean of a technical duplicate. (This figure is available in colour at JXB online.)

Fig. 6.

Time course accumulation of isoprenoid- and terpenoid-related transcripts in response to mechanical wounding in wild-type plantlets. Samples are the same as in Fig. 2. The black-filled shapes indicate fold changes (relative to controls) that are statistically significant according to Student's test at 0.05 (*), 0.01 (**), or 0.001 (***). ns, non significant. (This figure is available in colour at JXB online.)