Figure 1.
Sequence analysis of the PtrMYB216 cDNA.
(A) Sequence similarity of the N-terminal region of predicted PtrMYB216 including the conserved R2R3 DNA-binding domains with other MYB transcription factors. The R2R3 MYB domain is underlined. Identical and similar amino acid residues are shaded with black and gray, respectively. (B) Phylogenetic analysis of PtrMYB216 together with other MYBs involved in regulation of secondary wall biosynthesis or phenylpropanoid metabolism. Phylogenetic analysis was performed using the neighbor-joining method using MEGA version 4. Bar, 0.1 substitutions per site. Additional sequences include Arabidopsis thaliana (AtMYB26, At3g13890; AtMYB46, At5g12870; AtMYB58, At1g16490; AtMYB61, At1g09540; AtMYB63, At1g79180; AtMYB83, At3g08500; AtMYB85, At4g22680; AtMYB103, At1g63910), Zea mays (ZmMYB31, CAJ42202; ZmMYB42, CAJ42204), Eucalyptus gunnii (EgMYB1, CAE09058; EgMYB2, AJ576023), Pinus taeda (PtMYB1, AY356372; PtMYB4, AY356371), Populus tremula × tremuloides (PttMYB21a, AJ567345), P. trichocarpa (PtrMYB216, POPTR_0013s00290.1).
Figure 2.
Expression patterns of PtoMYB216 in various tissues of P. tomentosa Carr.
Semi-quantitative RT-PCR analysis of PtoMYB216 expression in various tissues of P. tomentosa Carr. (B) Quantitative real-time PCR analysis of PtoMYB216 transcript levels in various tissues of P. tomentosa Carr. Actin expression was used as a control. Total RNA was isolated from xylem, phloem, root, stem, petiole, old leaf, young leaf.
Figure 3.
Expression analysis of PtoMYB216 gene promoters.
PtoMYB216 promoter-driven GUS construct was generated (A) and introduced into Arabidopsis thaliana. Transgenic seedlings were grown on MS media and assayed for GUS activity. GUS expression was observed in various tissues of PtoMYB216::GUS plants, including leaf (B), flower (C), root (E), silique (F) and cortex and in vascular bundles in the cross section of stem (D).
Figure 4.
Nuclear localization and transcriptional activity of PtoMYB216.
(A) Onion epidermis was transformed with 35S:PtoMYB216:GFP and 35S:GFP constructs by particle bombardment. GFP fluorescent images were examined with confocal microscope at 18 h after bombardment. The position of nuclei was confirmed by DAPI staining and bright-filed images were compared. (B) Transcriptional activation analysis of PtoMYB216 ORF fused with the GAL4 DNA-binding domain (GAL4BD) showing its ability to activate the expression of the Trp and α-Gal reporter genes in yeast.
Figure 5.
Ectopic expression of PtoMYB216 results in an increase of xylem secondary cell wall thickness.
(A) View of vascular tissues stained by Phloroglucinol-HCl in transverse sections of stem and petiole of the 35S:PtoMYB216 plants and the wild-type. (B) Quantitative real-time PCR analysis of PtoMYB216 transcript levels in stems of transgenic poplar plants. WT, the wild type; 2, 3, and 7, PtoMYB216-overexpressing lines. Error bars represent SE of three replications.
Table 1.
Lignin content in the stems of the wild type and transgenic poplar overexpressing PtoMYB216 as determined by Klason analysis.
Figure 6.
Gene expression analyses of lignin biosynthetic genes in transgenic plants overexpressing PtoMYB216.
Transcript accumulation was quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The quantitative differences of the expression of the tested genes between the wild type and the overexpression lines are statistically significant (P<0.05). Error bars represent SE of three biological replicates. (A) Overexpression of PtoMYB216 induces the expression of lignin biosynthetic genes. The genes selected for expression analysis were previously shown to be highly expressed in stems where lignified fibers and vessels are abundant [51]. PAL4, phenylalanine ammonia lyase 4; C4H2, cinnamate 4-hydroxylase 2; 4CL5, 4-coumarate CoA ligase 5; C3H3, p-coumarate 3-hydroxylase 3; CCoAOMT1, caffeoyl CoA 3-O-methyltransferase 1; CCR2, cinnamoyl CoA reductase 2; F5H2, ferulate 5-hydroxylase 2; COMT2, caffeic acid O-methyltransferase 2; CAD1, cinnamyl alcohol dehydrogenase 1. (B) Overexpression of PtoMYB216 induces the expression of secondary wall-associated cellulose synthase genes (CesA2B, CesA3A), xylan biosynthetic genes (GT43B and GT43D).