<p>Ultrathin stem sections were stained with lead citrate and uranyl acetate and visualized... more <p>Ultrathin stem sections were stained with lead citrate and uranyl acetate and visualized for vessel secondary wall structure under a transmission electron microscope. The numbers 1, 2 and 3 marked on vessel secondary walls denote the S1, S2 and S3 layers, respectively. Note the drastic alteration in the staining pattern of the S2 layer as well as disintegrated walls in the S2 layer (arrows) in <i>tbl33 esk1</i> (C) and <i>tbl32 tbl33 esk1</i> (D). Bar in (A) = 3 μm for (A) to (D).</p
<p>The inflorescence stems of 8-week-old wild type and <i>esk1</i> plants, 12-w... more <p>The inflorescence stems of 8-week-old wild type and <i>esk1</i> plants, 12-week-old <i>tbl33 esk1</i> plants, and 16-week-old <i>tbl32 tbl33 esk1</i> plants were used for extraction of cell wall residues and xylan. (A) Cell wall composition analysis revealed a reduction in the amounts of xylose and glucose in <i>tbl33 esk1</i> and <i>tbl32 tbl33 esk1</i> compared with the wild type and <i>esk1</i>. (B) Acetyl contents in DMSO-extracted xylans of the wild type and various mutants. Note the drastic reduction in the acetyl contents in <i>tbl32 esk1</i>, <i>tbl33 esk1</i>, and <i>tbl32 tbl33 esk1</i>. Error bars denote SD of the data from three separate pools of samples. Asterisks in (A) and (B) indicate statistically significant differences compared with the wild type (<i>p</i> < 0.001). (C) MALDI-TOF-MS analysis of xylooligomers generated by endoxylanase digestion of KOH-extracted xylan from the wild type (top panel), <i>tbl32 tbl33</i> (middle panel) and <i>tbl32 tbl33 esk1</i> (bottom panel). The ion peaks at <i>m/z</i> 745 and 759 are attributed to (GlcA)Xyl<sub>4</sub> and (MeGlcA)Xyl<sub>4</sub>, respectively. Those at <i>m/z</i> 767 and 781 correspond to the disodiated species of (GlcA)Xyl<sub>4</sub> and (MeGlcA)Xyl<sub>4</sub>, respectively. The ion at <i>m/z</i> 775 corresponds to (Gal-GlcA)Xyl<sub>3</sub> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146460#pone.0146460.ref038" target="_blank">38</a>].</p
<p>(A) The sites of T-DNA insertions in the <i>TBL32</i> and <i>TBL33<... more <p>(A) The sites of T-DNA insertions in the <i>TBL32</i> and <i>TBL33</i> genes. Filled boxes represent exons. (B) Morphology of 4-week-old seedlings of the wild type and various mutants. (C) Morphology of 8-week-old wild-type and mutant plants. Inset shows the images of a 12-week-old plant of <i>tbl33 esk1</i> and a 16-week-old plant of <i>tbl32 tbl33 esk1</i>. Note the extremely dwarfed plants of <i>tbl33 esk1</i> and <i>tbl32 tbl33 esk1</i>. (D) Measurement of inflorescence stem heights during different developmental stages in the wild type, <i>tbl32</i>, <i>tbl33</i>, and <i>tbl32 tbl33</i> mutants. (E) Stem strength measurement in the wild type and various mutants. Basal parts of mature stems were measured for their breaking force. Error bars in (D) and (E) are SD of measurements from 20 independent plants. Asterisks in (E) indicate statistically significant differences compared with the wild type (<i>p</i> < 0.001).</p
<p>(A) Diagram of an acetylated xylooligomer from wild-type Arabidopsis xylan. (B) The fing... more <p>(A) Diagram of an acetylated xylooligomer from wild-type Arabidopsis xylan. (B) The fingerprint regions of the <sup>1</sup>H NMR spectra of acetylated xylans from the wild type, <i>tbl32 tbl33</i>, <i>esk1</i>, <i>tbl32 esk1</i>, <i>tbl33 esk1</i>, and <i>tbl32 tbl33 esk1</i>. The resonances for non-acetylated (Xyl), 2-<i>O</i>-acetylated (Xyl-2Ac), 3-<i>O</i>-acetylated (Xyl-3Ac), 2,3-di-<i>O</i>-acetylated (Xyl-2,3Ac), 3-<i>O</i>-acetylated 2-<i>O</i>-GlcA-substituted xylosyl residues (Xyl-3Ac-2GlcA) and GlcA/MeGlcA are labeled. Note the loss of the resonances of Xyl-3Ac-2GlcA in <i>tbl32 tbl33</i> and <i>tbl32 tbl33 esk1</i>.</p
<p>Cross sections of stems of the wild type (A, D, G), PtrMYB2-OE (B, E, H) and PtrMYB21-OE... more <p>Cross sections of stems of the wild type (A, D, G), PtrMYB2-OE (B, E, H) and PtrMYB21-OE (C, F, I) were stained for lignin with phloroglucinol (A to C), cellulose with Calcofluor White (D to F) and xylan with the LM10 xylan antibody (G to I). Note the ectopic deposition of lignin, cellulose and xylan in the epidermis and some cortical cells (arrows) of PtrMYB2-OE and PtrMYB21-OE. co, cortex; ep, epidermis; if, interfascicular fiber; pf, phloem fiber; xy, xylem. bars = 67 µm.</p
<p>(A) Shown are the SMRE consensus sequence and eight SMRE variants. (B) EMSA showing that... more <p>(A) Shown are the SMRE consensus sequence and eight SMRE variants. (B) EMSA showing that PtrMYB3, PtrMYB20, PtrMYB2, and PtrMYB21 all bind to the eight SMRE sequences. MBP, maltose binding protein. Each biotin-labeled SMRE probe was incubated with fusion proteins and the bound probes were separated from the free ones, which were detected by the chemiluminescent method.</p
<p>Stems of the transgenic control (transformed with the empty vector only), PtrMYB3-OE and... more <p>Stems of the transgenic control (transformed with the empty vector only), PtrMYB3-OE and PtrMYB21-OE were sectioned and stained for lignin with phloroglucinol-HCl, xylan with the LM10 xylan antibody, and cellulose with Calcofluor White. (A) to (C) Lignin staining of stem sections showing ectopic lignin deposition in cortical cells (arrows) in PtrMYB3-OE (B) and PtrMYB21-OE (C) compared with the control (A). (D) to (F) Xylan staining of stem sections showing ectopic xylan deposition in cortical cells (arrows) in PtrMYB3-OE (E) and PtrMYB21-OE (F) compared with the control (D). (G) to (I) Cellulose staining of stem sections showing ectopic cellulose deposition in cortical cells (arrows) in PtrMYB3-OE (H) and PtrMYB21-OE (I) compared with the control (G). co, cortex; pf, phloem fiber; sx, secondary xylem. Bars = 228 µm.</p
<p>The bottom parts of 6-month-old transgenic poplar plants were sectioned for examination ... more <p>The bottom parts of 6-month-old transgenic poplar plants were sectioned for examination of wood anatomy. The control is transgenic plants transformed with the empty vector only. (A) to (C) Toluidine blue-stained wood sections showing thinner secondary walls in xylary fibers and deformed vessel morphology in PtrMYB3-DR (B) and PtrMYB21-DR (C) compared with the control (A). (D) to (F) Transmission electron microscopy of wood sections showing reduced wall thickness in xylary fibers in PtrMYB3-DR (E) and PtrMYB21-DR (F) compared with the control (D). ve, vessel; xf, xylary fiber. Bar in (A) = 94 µm for (A) to (C), and bar in (D) = 4.9 µm for (D) to (F).</p
<p>The bottom internodes of stems and the root-hypocotyls of 8-week-old wild-type and <i... more <p>The bottom internodes of stems and the root-hypocotyls of 8-week-old wild-type and <i>esk1</i> plants, 12-week-old <i>tbl33 esk1</i> plants, and 16-week-old <i>tbl32 tbl33 esk1</i> plants were sectioned for visualization of anatomical structures. (A) to (D) Cross sections of stem xylem bundles showing vessels (arrows) with a mild deformation in <i>esk1</i> (B) and a severe deformation in <i>tbl33 esk1</i> (C) and <i>tbl32 tbl33 esk1</i> (D) compared with the wild type (A). (E) to (H) Cross sections of stem interfascicular regions showing defective secondary wall thickening in <i>esk1</i> (F), <i>tbl33 esk1</i> (G), and <i>tbl32 tbl33 esk1</i> (H) compared with the wild type (E). (I) to (L) Cross sections of root-hypocotyls showing vessels (arrows) with a mild deformation in <i>esk1</i> (J) and a severe deformation in <i>tbl33 esk1</i> (K) and <i>tbl32 tbl33 esk1</i> (L) compared with the wild type (I). if, interfascicular fiber; ph, phloem; sx, secondary xylem; xy, xylem. Bar in (A) = 68 μm for (A) to (L).</p
<p>(A) Phylogenetic relationship of Arabidopsis MYB46/MYB83 and their orthologs from poplar... more <p>(A) Phylogenetic relationship of Arabidopsis MYB46/MYB83 and their orthologs from poplar (<i>Populus trichocarpa</i>; PtrMYB2/3/20/21) and other plants, including <i>Eucalyptus</i> (<i>Eucalyptus grandis</i>; EgMYB2); Pine (<i>Pinus taeda</i>; PtMYB4), grapevine (<i>Vitis vinifera</i>; VvMYB46), alfalfa (<i>Medicago truncatula</i>; MtMYB46), soybean (<i>Glycine max</i>; GmMYB46), rice (<i>Oryza sativa</i>; OsMYB46), maize (<i>Zea mays</i>; ZmMYB46), sorghum (<i>Sorghum bicolor</i>; SbMYB46), barley (<i>Hordeum vulgare</i>; HvMYB46), and brachypodium (<i>Brachypodium distachyon</i>; BdMYB46). The phylogenetic tree was constructed with the neighbor-joining algorithm using PHYLIP and displayed using the TREEVIEW program. Bootstrap values are shown in percentages at the nodes. MYB58, MYB63 and their poplar homologs (PtrMYB28 and PtrMYB192) are included as the outgroup. (B) Complementation of <i>myb46 myb83</i> by PtrMYB2 and PtrMYB21. Upper panel shows four-week-old seedlings of the Arabidopsis <i>myb46 myb83</i> double mutant (arrow; higher magnification of <i>myb46 myb83</i> in inset), the <i>myb46 myb83</i> mutant expressing PtrMYB2 (+PtrMYB2), the <i>myb46 myb83</i> mutant expressing PtrMYB21 (+PtrMYB21), and the wild type. The lower panel shows secondary wall thickening in leaf veins of corresponding plants displayed above. Note that the vein in the <i>myb46 myb83</i> mutant has little secondary wall thickening, which is rescued by the expression of PtrMYB2 or PtrMYB21.</p
<p>(A) EMSA showing the binding of the eight SMRE sequences by EgMYB2 and PtMYB4. MBP and f... more <p>(A) EMSA showing the binding of the eight SMRE sequences by EgMYB2 and PtMYB4. MBP and fusion proteins (EgMYB2 and PtMYB4) were incubated with biotin-labeled SMRE probes and the bound probes were separated from the free ones, which were detected by the chemiluminescent method. (B) Transactivation analysis showing the activation of the SMRE-driven GUS reporter gene by EgMYB2 and PtMYB4 (lower panel). The reporter and effector constructs (upper panel) were co-transfected into Arabidopsis leaf protoplasts and after incubation, the transfected protoplasts were lysed and analyzed for the GUS activity. The control is the GUS activity in protoplasts transfected with the reporter construct and an empty effector construct without EgMYB2 or PtMYB4 and taken as 1. Error bars are the SE of three biological replicates.</p
<p>Three-week-old transgenic plants expressing <i>PtrMYB2</i> or <i>PtrMY... more <p>Three-week-old transgenic plants expressing <i>PtrMYB2</i> or <i>PtrMYB21</i> driven by the CaMV 35S promoter was examined for induction of secondary wall biosynthetic genes and ectopic deposition of secondary wall components. Bar in (C) = 64 µm for (C) to (H) and bar in (I) = 60 µm for (I) to (N). (A) Three-day-old seedlings of the wild type (left), a PtrMYB2 overexpressor (PtrMYB2-OE; middle), and a PtrMYB21 overexpressor (PtrMYB21-OE; right). Note the upward curly leaves in PtrMYB2-OE and PtrMYB21-OE. (B) Quantitative PCR analysis showing the induction of expression of secondary wall biosynthetic genes for cellulose (<i>CesA4</i>, <i>CesA7</i> and <i>CesA8</i>), xylan (<i>FRA8</i>, <i>IRX8</i> and <i>IRX9</i>) and lignin (<i>4CL1</i> and <i>CCoAOMT1</i>). The expression level of genes of interest in the wild type is set to 1. Error bars denote SE of three biological replicates. (C) and (D) Differential interference contrast (DIC) image (C) and lignin autofluorescence image (D) of the epidermis of a wild-type leaf. Note the low lignin signal in the inner wall of guard cells (arrows). (E) and (F) DIC image (E) and lignin autofluorescence image (F) of the leaf epidermis of a PtrMYB2 overexpressor showing ectopic wall thickening and lignin signal (arrowheads), respectively. (G) and (H) DIC image (G) and lignin autofluorescence image (H) of the leaf epidermis of a PtrMYB21 overexpressor showing ectopic wall thickening and lignin signal (arrowheads), respectively. (I) to (K) Sections of leaves of the wild type (I), PtrMYB2-OE (J) and PtrMYB21-OE (K) stained for cellulose with Calcofluor White. Note the strong signal for cellulose staining in the epidermal walls (arrows) of PtrMYB2-OE and PtrMYB21-OE. (L) to (N) Sections of leaves of the wild type (L), PtrMYB2-OE (M) and PtrMYB21-OE (N) stained for xylan with the LM10 xylan antibody. Note the strong signal for xylan staining in the epidermal walls (arrows) of PtrMYB2-OE and PtrMYB21-OE.</p
<p>(A) Diagrams of the effector and reporter constructs used for the transactivation analys... more <p>(A) Diagrams of the effector and reporter constructs used for the transactivation analysis. NosT, nopaline synthase terminator. (B) Transactivation analysis showing the activation by poplar MYB master switches of the GUS reporter gene driven by various secondary wall biosynthetic gene promoters. The effector and reporter constructs were cotransfected into Arabidopsis leaf protoplasts and after incubation, the transfected protoplasts were used for GUS activity assay. The GUS activity in protoplasts transfected with the reporter construct and an effector construct without MYB genes was set to 1. Error bars denote the SE of three biological replicates.</p
<p>(A) Diagrams of the effector and reporter constructs used for the transactivation analys... more <p>(A) Diagrams of the effector and reporter constructs used for the transactivation analysis. 3xSMRE, three copies of the SMRE sequence. (B) Transactivation analysis showing that PtrMYBs effectively activated the expression of the SMRE-driven GUS reporter gene. The reporter and effector constructs (A) were co-transfected into Arabidopsis leaf protoplasts and after incubation, the transfected protoplasts were lysed and analyzed for the GUS activity. The control is the GUS activity in protoplasts transfected with the reporter construct and an empty effector construct without PtrMYBs and taken as 1. Error bars are the SE of three biological replicates.</p
<p>The bottom internodes of stems of 8-week-old wild-type and <i>esk1</i> plant... more <p>The bottom internodes of stems of 8-week-old wild-type and <i>esk1</i> plants, 12-week-old <i>tbl33 esk1</i> plants, and 16-week-old <i>tbl32 tbl33 esk1</i> plants were sectioned for visualization of walls of interfascicular fibers and xylem vessels. (A) to (D) Cross sections of interfascicular fibers showing defective secondary wall thickening in <i>esk1</i> (B), <i>tbl33 esk1</i> (C), and <i>tbl32 tbl33 esk1</i> (D) compared with the wild type (A). (E) to (H) Cross sections of xylem cells showing various degrees of deformation in vessels in <i>esk1</i> (F), <i>tbl33 esk1</i> (G), and <i>tbl32 tbl33 esk1</i> (H) compared with the wild type (E). ve, vessel; xf, xylary fiber. Bar in (A) = 10.5 μm for (A) to (H).</p
<p>Arabidopsis leaf protoplasts expressing fluorescent protein-tagged fusion proteins were ... more <p>Arabidopsis leaf protoplasts expressing fluorescent protein-tagged fusion proteins were visualized for fluorescent signals with a laser confocal microscope. (A) TBL32 and TBL33 are type II membrane proteins based on the TMHMM2.0 program. Outside, the noncytoplasmic side of the membrane (Golgi); inside, the cytoplasmic side of the membrane. (B) An Arabidopsis protoplast expressing YFP alone showed the distribution of the signals throughout the cytoplasm. (C) to (F) An Arabidopsis protoplast (C) co-expressing TBL32-YFP (D) and the Golgi-localized FRA8-CFP (E). Note the overlap of the signals of TBL32-YFP and FRA8-CFP (F). (G) to (J) An Arabidopsis protoplast (G) co-expressing TBL33-YFP (H) and FRA8-CFP (I). Note the overlap of the signals of TBL33-YFP and FRA8-CFP (J). Bars in (B) to (J) = 12 μm.</p
<p>DMSO-extracted xylans were digested with endoxylanase and subject to MALDI-TOF MS. The m... more <p>DMSO-extracted xylans were digested with endoxylanase and subject to MALDI-TOF MS. The major ion peaks of masses are indicated and their xylooligomer structures are listed below. Xyl<sub>n</sub>(GlcA)<sub>n</sub>(Ac)<sub>n</sub> denotes a xylooligomer (with n number of xylosyl residues) substituted with n number of GlcA and n number of acetyl groups. <i>m/z</i> 743, Xyl<sub>5</sub>(Ac); <i>m/z</i> 745, Xyl<sub>4</sub>(GlcA); <i>m/z</i> 759, Xyl<sub>4</sub>(MeGlcA); <i>m/z</i> 787, Xyl<sub>4</sub>(GlcA)(Ac); <i>m/z</i> 801, Xyl<sub>4</sub>(MeGlcA)(Ac); <i>m/z</i> 817, Xyl<sub>3</sub>(MeGlcA)<sub>2</sub>; <i>m/z</i> 843, Xyl<sub>4</sub>(MeGlcA)(Ac)<sub>2</sub>; <i>m/z</i> 877, Xyl<sub>5</sub>(GlcA); <i>m/z</i> 891, Xyl<sub>5</sub>(MeGlcA); <i>m/z</i> 919, Xyl<sub>5</sub>(GlcA)(Ac); <i>m/z</i> 975, Xyl<sub>5</sub>(MeGlcA)(Ac)<sub>2</sub>; <i>m/z</i> 1017, Xyl<sub>5</sub>(MeGlcA)(Ac)<sub>3</sub>; <i>m/z</i> 1051, Xyl<sub>6</sub>(GlcA)(Ac); <i>m/z</i> 1065, Xyl<sub>6</sub>(MeGlcA)(Ac); <i>m/z</i> 1093; Xyl<sub>6</sub>(GlcA)(Ac)<sub>2</sub>; <i>m/z</i> 1107; Xyl<sub>6</sub>(MeGlcA)(Ac)<sub>2</sub>; <i>m/z</i> 1123, Xyl<sub>5</sub>(MeGlcA)<sub>2</sub>(Ac); <i>m/z</i> 1135, Xyl<sub>6</sub>(GlcA)(Ac)<sub>3</sub>; <i>m/z</i> 1149, Xyl<sub>6</sub>(MeGlcA)(Ac)<sub>3</sub>; <i>m/z</i> 1165, Xyl<sub>5</sub>(MeGlcA)<sub>2</sub>(Ac)<sub>2</sub>; <i>m/z</i> 1183, Xyl<sub>7</sub>(GlcA)(Ac); <i>m/z</i> 1197, Xyl<sub>7</sub>(MeGlcA)(Ac); <i>m/z</i> 1225, Xyl<sub>7</sub>(GlcA)(Ac)<sub>2</sub>; <i>m/z</i> 1241, Xyl<sub>6</sub>(GlcA)(MeGlcA)(Ac); <i>m/z</i> 1267, Xyl<sub>7</sub>(GlcA)(Ac)<sub>3</sub>; <i>m/z</i> 1283, Xyl<sub>6</sub>(GlcA)(MeGlcA)(Ac)<sub>2</sub>; <i>m/z</i> 1357, Xyl<sub>8</sub>(GlcA)(Ac)<sub>2</sub>. Note the increase in the abundance of ions at <i>m/z</i> 745, 759, 877, and 891 (marked in red) corresponding to non-acetylated xylooligomers in <i>tbl32 tbl33</i>, <i>tbl32 esk1</i>, <i>tbl33 esk1</i>, and <i>tbl32 tbl33 esk1</i>.</p
<p>The number shown at the left of each sequence denotes the position of the first nucleoti... more <p>The number shown at the left of each sequence denotes the position of the first nucleotide relative to the start codon. The plus or minus symbol at the right indicates the sequence from the forward or reverse strand of DNA, respectively.</p
<p>Ultrathin stem sections were stained with lead citrate and uranyl acetate and visualized... more <p>Ultrathin stem sections were stained with lead citrate and uranyl acetate and visualized for vessel secondary wall structure under a transmission electron microscope. The numbers 1, 2 and 3 marked on vessel secondary walls denote the S1, S2 and S3 layers, respectively. Note the drastic alteration in the staining pattern of the S2 layer as well as disintegrated walls in the S2 layer (arrows) in <i>tbl33 esk1</i> (C) and <i>tbl32 tbl33 esk1</i> (D). Bar in (A) = 3 μm for (A) to (D).</p
<p>The inflorescence stems of 8-week-old wild type and <i>esk1</i> plants, 12-w... more <p>The inflorescence stems of 8-week-old wild type and <i>esk1</i> plants, 12-week-old <i>tbl33 esk1</i> plants, and 16-week-old <i>tbl32 tbl33 esk1</i> plants were used for extraction of cell wall residues and xylan. (A) Cell wall composition analysis revealed a reduction in the amounts of xylose and glucose in <i>tbl33 esk1</i> and <i>tbl32 tbl33 esk1</i> compared with the wild type and <i>esk1</i>. (B) Acetyl contents in DMSO-extracted xylans of the wild type and various mutants. Note the drastic reduction in the acetyl contents in <i>tbl32 esk1</i>, <i>tbl33 esk1</i>, and <i>tbl32 tbl33 esk1</i>. Error bars denote SD of the data from three separate pools of samples. Asterisks in (A) and (B) indicate statistically significant differences compared with the wild type (<i>p</i> < 0.001). (C) MALDI-TOF-MS analysis of xylooligomers generated by endoxylanase digestion of KOH-extracted xylan from the wild type (top panel), <i>tbl32 tbl33</i> (middle panel) and <i>tbl32 tbl33 esk1</i> (bottom panel). The ion peaks at <i>m/z</i> 745 and 759 are attributed to (GlcA)Xyl<sub>4</sub> and (MeGlcA)Xyl<sub>4</sub>, respectively. Those at <i>m/z</i> 767 and 781 correspond to the disodiated species of (GlcA)Xyl<sub>4</sub> and (MeGlcA)Xyl<sub>4</sub>, respectively. The ion at <i>m/z</i> 775 corresponds to (Gal-GlcA)Xyl<sub>3</sub> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146460#pone.0146460.ref038" target="_blank">38</a>].</p
<p>(A) The sites of T-DNA insertions in the <i>TBL32</i> and <i>TBL33<... more <p>(A) The sites of T-DNA insertions in the <i>TBL32</i> and <i>TBL33</i> genes. Filled boxes represent exons. (B) Morphology of 4-week-old seedlings of the wild type and various mutants. (C) Morphology of 8-week-old wild-type and mutant plants. Inset shows the images of a 12-week-old plant of <i>tbl33 esk1</i> and a 16-week-old plant of <i>tbl32 tbl33 esk1</i>. Note the extremely dwarfed plants of <i>tbl33 esk1</i> and <i>tbl32 tbl33 esk1</i>. (D) Measurement of inflorescence stem heights during different developmental stages in the wild type, <i>tbl32</i>, <i>tbl33</i>, and <i>tbl32 tbl33</i> mutants. (E) Stem strength measurement in the wild type and various mutants. Basal parts of mature stems were measured for their breaking force. Error bars in (D) and (E) are SD of measurements from 20 independent plants. Asterisks in (E) indicate statistically significant differences compared with the wild type (<i>p</i> < 0.001).</p
<p>(A) Diagram of an acetylated xylooligomer from wild-type Arabidopsis xylan. (B) The fing... more <p>(A) Diagram of an acetylated xylooligomer from wild-type Arabidopsis xylan. (B) The fingerprint regions of the <sup>1</sup>H NMR spectra of acetylated xylans from the wild type, <i>tbl32 tbl33</i>, <i>esk1</i>, <i>tbl32 esk1</i>, <i>tbl33 esk1</i>, and <i>tbl32 tbl33 esk1</i>. The resonances for non-acetylated (Xyl), 2-<i>O</i>-acetylated (Xyl-2Ac), 3-<i>O</i>-acetylated (Xyl-3Ac), 2,3-di-<i>O</i>-acetylated (Xyl-2,3Ac), 3-<i>O</i>-acetylated 2-<i>O</i>-GlcA-substituted xylosyl residues (Xyl-3Ac-2GlcA) and GlcA/MeGlcA are labeled. Note the loss of the resonances of Xyl-3Ac-2GlcA in <i>tbl32 tbl33</i> and <i>tbl32 tbl33 esk1</i>.</p
<p>Cross sections of stems of the wild type (A, D, G), PtrMYB2-OE (B, E, H) and PtrMYB21-OE... more <p>Cross sections of stems of the wild type (A, D, G), PtrMYB2-OE (B, E, H) and PtrMYB21-OE (C, F, I) were stained for lignin with phloroglucinol (A to C), cellulose with Calcofluor White (D to F) and xylan with the LM10 xylan antibody (G to I). Note the ectopic deposition of lignin, cellulose and xylan in the epidermis and some cortical cells (arrows) of PtrMYB2-OE and PtrMYB21-OE. co, cortex; ep, epidermis; if, interfascicular fiber; pf, phloem fiber; xy, xylem. bars = 67 µm.</p
<p>(A) Shown are the SMRE consensus sequence and eight SMRE variants. (B) EMSA showing that... more <p>(A) Shown are the SMRE consensus sequence and eight SMRE variants. (B) EMSA showing that PtrMYB3, PtrMYB20, PtrMYB2, and PtrMYB21 all bind to the eight SMRE sequences. MBP, maltose binding protein. Each biotin-labeled SMRE probe was incubated with fusion proteins and the bound probes were separated from the free ones, which were detected by the chemiluminescent method.</p
<p>Stems of the transgenic control (transformed with the empty vector only), PtrMYB3-OE and... more <p>Stems of the transgenic control (transformed with the empty vector only), PtrMYB3-OE and PtrMYB21-OE were sectioned and stained for lignin with phloroglucinol-HCl, xylan with the LM10 xylan antibody, and cellulose with Calcofluor White. (A) to (C) Lignin staining of stem sections showing ectopic lignin deposition in cortical cells (arrows) in PtrMYB3-OE (B) and PtrMYB21-OE (C) compared with the control (A). (D) to (F) Xylan staining of stem sections showing ectopic xylan deposition in cortical cells (arrows) in PtrMYB3-OE (E) and PtrMYB21-OE (F) compared with the control (D). (G) to (I) Cellulose staining of stem sections showing ectopic cellulose deposition in cortical cells (arrows) in PtrMYB3-OE (H) and PtrMYB21-OE (I) compared with the control (G). co, cortex; pf, phloem fiber; sx, secondary xylem. Bars = 228 µm.</p
<p>The bottom parts of 6-month-old transgenic poplar plants were sectioned for examination ... more <p>The bottom parts of 6-month-old transgenic poplar plants were sectioned for examination of wood anatomy. The control is transgenic plants transformed with the empty vector only. (A) to (C) Toluidine blue-stained wood sections showing thinner secondary walls in xylary fibers and deformed vessel morphology in PtrMYB3-DR (B) and PtrMYB21-DR (C) compared with the control (A). (D) to (F) Transmission electron microscopy of wood sections showing reduced wall thickness in xylary fibers in PtrMYB3-DR (E) and PtrMYB21-DR (F) compared with the control (D). ve, vessel; xf, xylary fiber. Bar in (A) = 94 µm for (A) to (C), and bar in (D) = 4.9 µm for (D) to (F).</p
<p>The bottom internodes of stems and the root-hypocotyls of 8-week-old wild-type and <i... more <p>The bottom internodes of stems and the root-hypocotyls of 8-week-old wild-type and <i>esk1</i> plants, 12-week-old <i>tbl33 esk1</i> plants, and 16-week-old <i>tbl32 tbl33 esk1</i> plants were sectioned for visualization of anatomical structures. (A) to (D) Cross sections of stem xylem bundles showing vessels (arrows) with a mild deformation in <i>esk1</i> (B) and a severe deformation in <i>tbl33 esk1</i> (C) and <i>tbl32 tbl33 esk1</i> (D) compared with the wild type (A). (E) to (H) Cross sections of stem interfascicular regions showing defective secondary wall thickening in <i>esk1</i> (F), <i>tbl33 esk1</i> (G), and <i>tbl32 tbl33 esk1</i> (H) compared with the wild type (E). (I) to (L) Cross sections of root-hypocotyls showing vessels (arrows) with a mild deformation in <i>esk1</i> (J) and a severe deformation in <i>tbl33 esk1</i> (K) and <i>tbl32 tbl33 esk1</i> (L) compared with the wild type (I). if, interfascicular fiber; ph, phloem; sx, secondary xylem; xy, xylem. Bar in (A) = 68 μm for (A) to (L).</p
<p>(A) Phylogenetic relationship of Arabidopsis MYB46/MYB83 and their orthologs from poplar... more <p>(A) Phylogenetic relationship of Arabidopsis MYB46/MYB83 and their orthologs from poplar (<i>Populus trichocarpa</i>; PtrMYB2/3/20/21) and other plants, including <i>Eucalyptus</i> (<i>Eucalyptus grandis</i>; EgMYB2); Pine (<i>Pinus taeda</i>; PtMYB4), grapevine (<i>Vitis vinifera</i>; VvMYB46), alfalfa (<i>Medicago truncatula</i>; MtMYB46), soybean (<i>Glycine max</i>; GmMYB46), rice (<i>Oryza sativa</i>; OsMYB46), maize (<i>Zea mays</i>; ZmMYB46), sorghum (<i>Sorghum bicolor</i>; SbMYB46), barley (<i>Hordeum vulgare</i>; HvMYB46), and brachypodium (<i>Brachypodium distachyon</i>; BdMYB46). The phylogenetic tree was constructed with the neighbor-joining algorithm using PHYLIP and displayed using the TREEVIEW program. Bootstrap values are shown in percentages at the nodes. MYB58, MYB63 and their poplar homologs (PtrMYB28 and PtrMYB192) are included as the outgroup. (B) Complementation of <i>myb46 myb83</i> by PtrMYB2 and PtrMYB21. Upper panel shows four-week-old seedlings of the Arabidopsis <i>myb46 myb83</i> double mutant (arrow; higher magnification of <i>myb46 myb83</i> in inset), the <i>myb46 myb83</i> mutant expressing PtrMYB2 (+PtrMYB2), the <i>myb46 myb83</i> mutant expressing PtrMYB21 (+PtrMYB21), and the wild type. The lower panel shows secondary wall thickening in leaf veins of corresponding plants displayed above. Note that the vein in the <i>myb46 myb83</i> mutant has little secondary wall thickening, which is rescued by the expression of PtrMYB2 or PtrMYB21.</p
<p>(A) EMSA showing the binding of the eight SMRE sequences by EgMYB2 and PtMYB4. MBP and f... more <p>(A) EMSA showing the binding of the eight SMRE sequences by EgMYB2 and PtMYB4. MBP and fusion proteins (EgMYB2 and PtMYB4) were incubated with biotin-labeled SMRE probes and the bound probes were separated from the free ones, which were detected by the chemiluminescent method. (B) Transactivation analysis showing the activation of the SMRE-driven GUS reporter gene by EgMYB2 and PtMYB4 (lower panel). The reporter and effector constructs (upper panel) were co-transfected into Arabidopsis leaf protoplasts and after incubation, the transfected protoplasts were lysed and analyzed for the GUS activity. The control is the GUS activity in protoplasts transfected with the reporter construct and an empty effector construct without EgMYB2 or PtMYB4 and taken as 1. Error bars are the SE of three biological replicates.</p
<p>Three-week-old transgenic plants expressing <i>PtrMYB2</i> or <i>PtrMY... more <p>Three-week-old transgenic plants expressing <i>PtrMYB2</i> or <i>PtrMYB21</i> driven by the CaMV 35S promoter was examined for induction of secondary wall biosynthetic genes and ectopic deposition of secondary wall components. Bar in (C) = 64 µm for (C) to (H) and bar in (I) = 60 µm for (I) to (N). (A) Three-day-old seedlings of the wild type (left), a PtrMYB2 overexpressor (PtrMYB2-OE; middle), and a PtrMYB21 overexpressor (PtrMYB21-OE; right). Note the upward curly leaves in PtrMYB2-OE and PtrMYB21-OE. (B) Quantitative PCR analysis showing the induction of expression of secondary wall biosynthetic genes for cellulose (<i>CesA4</i>, <i>CesA7</i> and <i>CesA8</i>), xylan (<i>FRA8</i>, <i>IRX8</i> and <i>IRX9</i>) and lignin (<i>4CL1</i> and <i>CCoAOMT1</i>). The expression level of genes of interest in the wild type is set to 1. Error bars denote SE of three biological replicates. (C) and (D) Differential interference contrast (DIC) image (C) and lignin autofluorescence image (D) of the epidermis of a wild-type leaf. Note the low lignin signal in the inner wall of guard cells (arrows). (E) and (F) DIC image (E) and lignin autofluorescence image (F) of the leaf epidermis of a PtrMYB2 overexpressor showing ectopic wall thickening and lignin signal (arrowheads), respectively. (G) and (H) DIC image (G) and lignin autofluorescence image (H) of the leaf epidermis of a PtrMYB21 overexpressor showing ectopic wall thickening and lignin signal (arrowheads), respectively. (I) to (K) Sections of leaves of the wild type (I), PtrMYB2-OE (J) and PtrMYB21-OE (K) stained for cellulose with Calcofluor White. Note the strong signal for cellulose staining in the epidermal walls (arrows) of PtrMYB2-OE and PtrMYB21-OE. (L) to (N) Sections of leaves of the wild type (L), PtrMYB2-OE (M) and PtrMYB21-OE (N) stained for xylan with the LM10 xylan antibody. Note the strong signal for xylan staining in the epidermal walls (arrows) of PtrMYB2-OE and PtrMYB21-OE.</p
<p>(A) Diagrams of the effector and reporter constructs used for the transactivation analys... more <p>(A) Diagrams of the effector and reporter constructs used for the transactivation analysis. NosT, nopaline synthase terminator. (B) Transactivation analysis showing the activation by poplar MYB master switches of the GUS reporter gene driven by various secondary wall biosynthetic gene promoters. The effector and reporter constructs were cotransfected into Arabidopsis leaf protoplasts and after incubation, the transfected protoplasts were used for GUS activity assay. The GUS activity in protoplasts transfected with the reporter construct and an effector construct without MYB genes was set to 1. Error bars denote the SE of three biological replicates.</p
<p>(A) Diagrams of the effector and reporter constructs used for the transactivation analys... more <p>(A) Diagrams of the effector and reporter constructs used for the transactivation analysis. 3xSMRE, three copies of the SMRE sequence. (B) Transactivation analysis showing that PtrMYBs effectively activated the expression of the SMRE-driven GUS reporter gene. The reporter and effector constructs (A) were co-transfected into Arabidopsis leaf protoplasts and after incubation, the transfected protoplasts were lysed and analyzed for the GUS activity. The control is the GUS activity in protoplasts transfected with the reporter construct and an empty effector construct without PtrMYBs and taken as 1. Error bars are the SE of three biological replicates.</p
<p>The bottom internodes of stems of 8-week-old wild-type and <i>esk1</i> plant... more <p>The bottom internodes of stems of 8-week-old wild-type and <i>esk1</i> plants, 12-week-old <i>tbl33 esk1</i> plants, and 16-week-old <i>tbl32 tbl33 esk1</i> plants were sectioned for visualization of walls of interfascicular fibers and xylem vessels. (A) to (D) Cross sections of interfascicular fibers showing defective secondary wall thickening in <i>esk1</i> (B), <i>tbl33 esk1</i> (C), and <i>tbl32 tbl33 esk1</i> (D) compared with the wild type (A). (E) to (H) Cross sections of xylem cells showing various degrees of deformation in vessels in <i>esk1</i> (F), <i>tbl33 esk1</i> (G), and <i>tbl32 tbl33 esk1</i> (H) compared with the wild type (E). ve, vessel; xf, xylary fiber. Bar in (A) = 10.5 μm for (A) to (H).</p
<p>Arabidopsis leaf protoplasts expressing fluorescent protein-tagged fusion proteins were ... more <p>Arabidopsis leaf protoplasts expressing fluorescent protein-tagged fusion proteins were visualized for fluorescent signals with a laser confocal microscope. (A) TBL32 and TBL33 are type II membrane proteins based on the TMHMM2.0 program. Outside, the noncytoplasmic side of the membrane (Golgi); inside, the cytoplasmic side of the membrane. (B) An Arabidopsis protoplast expressing YFP alone showed the distribution of the signals throughout the cytoplasm. (C) to (F) An Arabidopsis protoplast (C) co-expressing TBL32-YFP (D) and the Golgi-localized FRA8-CFP (E). Note the overlap of the signals of TBL32-YFP and FRA8-CFP (F). (G) to (J) An Arabidopsis protoplast (G) co-expressing TBL33-YFP (H) and FRA8-CFP (I). Note the overlap of the signals of TBL33-YFP and FRA8-CFP (J). Bars in (B) to (J) = 12 μm.</p
<p>DMSO-extracted xylans were digested with endoxylanase and subject to MALDI-TOF MS. The m... more <p>DMSO-extracted xylans were digested with endoxylanase and subject to MALDI-TOF MS. The major ion peaks of masses are indicated and their xylooligomer structures are listed below. Xyl<sub>n</sub>(GlcA)<sub>n</sub>(Ac)<sub>n</sub> denotes a xylooligomer (with n number of xylosyl residues) substituted with n number of GlcA and n number of acetyl groups. <i>m/z</i> 743, Xyl<sub>5</sub>(Ac); <i>m/z</i> 745, Xyl<sub>4</sub>(GlcA); <i>m/z</i> 759, Xyl<sub>4</sub>(MeGlcA); <i>m/z</i> 787, Xyl<sub>4</sub>(GlcA)(Ac); <i>m/z</i> 801, Xyl<sub>4</sub>(MeGlcA)(Ac); <i>m/z</i> 817, Xyl<sub>3</sub>(MeGlcA)<sub>2</sub>; <i>m/z</i> 843, Xyl<sub>4</sub>(MeGlcA)(Ac)<sub>2</sub>; <i>m/z</i> 877, Xyl<sub>5</sub>(GlcA); <i>m/z</i> 891, Xyl<sub>5</sub>(MeGlcA); <i>m/z</i> 919, Xyl<sub>5</sub>(GlcA)(Ac); <i>m/z</i> 975, Xyl<sub>5</sub>(MeGlcA)(Ac)<sub>2</sub>; <i>m/z</i> 1017, Xyl<sub>5</sub>(MeGlcA)(Ac)<sub>3</sub>; <i>m/z</i> 1051, Xyl<sub>6</sub>(GlcA)(Ac); <i>m/z</i> 1065, Xyl<sub>6</sub>(MeGlcA)(Ac); <i>m/z</i> 1093; Xyl<sub>6</sub>(GlcA)(Ac)<sub>2</sub>; <i>m/z</i> 1107; Xyl<sub>6</sub>(MeGlcA)(Ac)<sub>2</sub>; <i>m/z</i> 1123, Xyl<sub>5</sub>(MeGlcA)<sub>2</sub>(Ac); <i>m/z</i> 1135, Xyl<sub>6</sub>(GlcA)(Ac)<sub>3</sub>; <i>m/z</i> 1149, Xyl<sub>6</sub>(MeGlcA)(Ac)<sub>3</sub>; <i>m/z</i> 1165, Xyl<sub>5</sub>(MeGlcA)<sub>2</sub>(Ac)<sub>2</sub>; <i>m/z</i> 1183, Xyl<sub>7</sub>(GlcA)(Ac); <i>m/z</i> 1197, Xyl<sub>7</sub>(MeGlcA)(Ac); <i>m/z</i> 1225, Xyl<sub>7</sub>(GlcA)(Ac)<sub>2</sub>; <i>m/z</i> 1241, Xyl<sub>6</sub>(GlcA)(MeGlcA)(Ac); <i>m/z</i> 1267, Xyl<sub>7</sub>(GlcA)(Ac)<sub>3</sub>; <i>m/z</i> 1283, Xyl<sub>6</sub>(GlcA)(MeGlcA)(Ac)<sub>2</sub>; <i>m/z</i> 1357, Xyl<sub>8</sub>(GlcA)(Ac)<sub>2</sub>. Note the increase in the abundance of ions at <i>m/z</i> 745, 759, 877, and 891 (marked in red) corresponding to non-acetylated xylooligomers in <i>tbl32 tbl33</i>, <i>tbl32 esk1</i>, <i>tbl33 esk1</i>, and <i>tbl32 tbl33 esk1</i>.</p
<p>The number shown at the left of each sequence denotes the position of the first nucleoti... more <p>The number shown at the left of each sequence denotes the position of the first nucleotide relative to the start codon. The plus or minus symbol at the right indicates the sequence from the forward or reverse strand of DNA, respectively.</p
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