The poplar MYB master switches bind to the SMRE site and activate the secondary wall biosynthetic program during wood formation.

Wood is mainly composed of secondary walls, which constitute the most abundant stored carbon produced by vascular plants. Understanding the molecular mechanisms controlling secondary wall deposition during wood formation is not only an important issue in plant biology but also critical for providing molecular tools to custom-design wood composition suited for diverse end uses. Past molecular and genetic studies have revealed a transcriptional network encompassing a group of wood-associated NAC and MYB transcription factors that are involved in the regulation of the secondary wall biosynthetic program during wood formation in poplar trees. Here, we report the functional characterization of poplar orthologs of MYB46 and MYB83 that are known to be master switches of secondary wall biosynthesis in Arabidopsis. In addition to the two previously-described PtrMYB3 and PtrMYB20, two other MYBs, PtrMYB2 and PtrMYB21, were shown to be MYB46/MYB83 orthologs by complementation and overexpression studies in Arabidopsis. The functional roles of these PtrMYBs in regulating secondary wall biosynthesis were further demonstrated in transgenic poplar plants showing an ectopic deposition of secondary walls in PtrMYB overexpressors and a reduction of secondary wall thickening in their dominant repressors. Furthermore, PtrMYB2/3/20/21 together with two other tree MYBs, the Eucalyptus EgMYB2 and the pine PtMYB4, were shown to differentially bind to and activate the eight variants of the 7-bp SMRE consensus sequence, composed of ACC(A/T)A(A/C)(T/C). Together, our results indicate that the tree MYBs, PtrMYB2/3/20/21, EgMYB2 and PtMYB4, are master transcriptional switches that activate the SMRE sites in the promoters of target genes and thereby regulate secondary wall biosynthesis during wood formation

Mutations of Arabidopsis TBL32 and TBL33 Affect Xylan Acetylation and Secondary Wall Deposition.

Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls

EgMYB2 and PtMYB4 bind to and activate the SMRE sequences.

<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

Overexpression of PtrMYB3 and PtrMYB21 causes ectopic deposition of secondary wall components in transgenic poplar stems.

<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

Reduction in secondary wall thickening and alteration in vessel morphology in the wood of transgenic poplar with dominant repression of PtrMYB3 (PtrMYB3-DR) and PtrMYB21 (PtrMYB21-DR).

<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

Identification of the SNBE sequences in the 1.5-kb promoters of of poplar cellulose and xylan biosynthetic genes based on the SMRE consensus sequence.

<p>Identification of the SNBE sequences in the 1.5-kb promoters of of poplar cellulose and xylan biosynthetic genes based on the SMRE consensus sequence.</p

Induction of ectopic deposition of secondary wall components in the epidermis of Arabidopsis stems by overexpression of PtrMYB2 and PtrMYB21.

<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

Identification of the SNBE sequences in the 1.5-kb promoters of PtrMYB2/3/20/21 genes based on the SNBE consensus sequence (A) and the SMRE sequences in the 1.5-kb promoters of poplar lignin biosynthetic genes based on the SMRE consensus sequence (B).

<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

EMSA of binding of PtrMYB3, PtrMYB20, PtrMYB2, and PtrMYB21 to the SMRE sequences.

<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