Dissection of the transcriptional program regulating secondary wall biosynthesis during wood formation in poplar

Abstract

Wood biomass is mainly made of secondary cell walls; hence, elucidation of the molecular mechanisms underlying the transcriptional regulation of secondary wall biosynthesis during wood formation will be instrumental to design strategies for genetic improvement of wood biomass. Here, we provide direct evidence demonstrating that the poplar (Populus trichocarpa) wood-associated NAC domain transcription factors (PtrWNDs) are master switches activating a suite of downstream transcription factors, and together, they are involved in the coordinated regulation of secondary wall biosynthesis during wood formation. We show that transgenic poplar plants with dominant repression of PtrWNDs functions exhibit a drastic reduction in secondary wall thickening in woody cells, and those with PtrWND overexpression result in ectopic deposition of secondary walls. Analysis of PtrWND2B overexpressors revealed up-regulation of the expression of a number of wood-associated transcription factors, the promoters of which were also activated by PtrWND6B and the Eucalyptus EgWND1. Transactivation analysis and electrophoretic mobility shift assay demonstrated that PtrWNDs and EgWND1 activated gene expression through direct binding to the secondary wall NAC-binding elements, which are present in the promoters of several wood-associated transcription factors and a number of genes involved in secondary wall biosynthesis and modification. The WND-regulated transcription factors PtrNAC150, PtrNAC156, PtrNAC157, PtrMYB18, PtrMYB74, PtrMYB75, PtrMYB121, PtrMYB128, PtrZF1, and PtrGATA8 were able to activate the promoter activities of the biosynthetic genes for all three major wood components. Our study has uncovered that the WND master switches together with a battery of their downstream transcription factors form a transcriptional network controlling secondary wall biosynthesis during wood formation.

Figure 1.

Effect of dominant repression of PtrWND2B and PtrWND6B on secondary wall thickening and vessel morphology in the wood of transgenic poplar. The full-length WND cDNAs fused in frame with the dominant EAR repression sequence were expressed in transgenic poplar plants. The bottom parts of 6-month-old plants were used for the examination of secondary walls in fibers and vessels. A, RT-PCR analysis showing the presence of the transcript of the PtrWND2B repressor (PtrWND2B-DR; left panel) or the PtrWND6B repressor (PtrWND6B-DR; right panel) in the stems of five independent representative transgenic lines. The control is a transgenic plant transformed with the empty vector only. The expression level of a poplar actin gene (PtrACTIN) was used as a control. B, Representative control plant (left) and transgenic poplar plant expressing the PtrWND2B repressor (middle) or the PtrWND6B repressor (right). C to E, Cross-sections of stems showing reduced wall thickness in xylary fibers and deformed vessel morphology (arrows) in PtrWND2B-DR (D) and PtrWND6B (E) compared with the control plant (C). F to H, Transmission electron micrographs of xylary fibers showing reduced wall thickness in PtrWND2B-DR (G) and PtrWND6B (H) compared with the control plant (F). ve, Vessel; xf, xylary fiber. Bar in C = 85 μm for C to E, and bar in F = 3.2 μm for F to H. [See online article for color version of this figure.]

Figure 2.

Presence of cellulose, xylan, and lignin in the ectopically deposited secondary walls in the stems of PtrWND overexpressors. The full-length PtrWND2B and PtrWND6B cDNAs driven by the CaMV 35S promoter were transformed into poplar, and the stems of the transgenic overexpressors were stained for lignin with phloroglucinol-HCl, for cellulose with Calcofluor White, and for xylan with the LM10 xylan monoclonal antibody. A, Real-time quantitative PCR analysis showing the overexpression of PtrWND2B (PtrWND2B-OE; left panel) and PtrWND6B (PtrWND6B-OE; right panel) in the stems of six representative transgenic lines. The expression level of PtrWND2B and PtrWND6B in the control plant was set to 1. Error bars represent the se of three replicates. B, Representative control plant (left) and transgenic poplar plant overexpressing PtrWND2B (middle) or PtrWND6B (right). C, Leaf morphology of the control plant (left) and a transgenic poplar plant expressing PtrWND2B (middle and right). D to F, Phloroglucinol-HCl staining of stem sections showing intensive lignin staining in the walls of some cortical cells (arrows) in addition to the phloem fibers and secondary xylem cell walls in the PtrWND2B (E) and PtrWND6B (F) overexpressors compared with the control (D). G to I, Calcofluor White staining of stem sections showing intensive cellulose staining in the walls of some cortical cells (arrows) in addition to the phloem fibers and secondary xylem cell walls in the PtrWND2B (H) and PtrWND6B (I) overexpressors compared with the control (G). J to L, Stem sections probed with the LM10 xylan monoclonal antibody showing intensive xylan staining in the walls of some cortical cells (arrows) in addition to the phloem fibers and secondary xylem cell walls in the PtrWND2B (K) and PtrWND6B (L) overexpressors compared with the control (J). co, Cortex; pf, phloem fiber; sx, secondary xylem. Bars in D to L = 155 μm.

Figure 3.

Excess overexpression of PtrWND2B in transgenic poplar results in curly leaves with ectopic secondary wall deposition. The leaves of transgenic poplar seedlings were examined for ectopic deposition of lignin, cellulose, and xylan. A, Representative control poplar seedling. B, PtrWND2B overexpressor (PtrWND2B-OE) seedling with curly leaves. C, Closeup view of the control (left) and the curly PtrWND2B-OE (right) leaves. D, Real-time quantitative PCR analysis showing the excess overexpression of PtrWND2B in the seedlings of four independent representative transgenic lines (left panel) and the activation of secondary wall biosynthetic genes (PtrCesA4/7/8/17/18 for cellulose, PtrGT43B and PtrGT47C for xylan, and PtrCCoAOMT1 and PtrCOMT2 for lignin) and two primary wall cellulose synthase genes (PtrCesA6 and PtrCesA15; right panel). The expression of PtrWND2B or cell wall biosynthetic genes in the control was set to 1. Error bars represent the se of three biological replicates. E and F, Differential interference contrast (E) and lignin autofluorescence (F) images of a control leaf showing the lignified secondary wall thickening in veins (arrows). G and H, Differential interference contrast (G) and lignin autofluorescence (H) images of leaf mesophyll cells of PtrWND2B overexpressors showing ectopic deposition of secondary walls and lignin. I to K, Sections of the control leaves (I) stained for xylan (J) and cellulose (K). L to N, Sections of PtrWND2B overexpressor leaves (L) showing ectopic deposition of xylan (M) and secondary wall cellulose (N) in the mesophyll cells (arrows). Bars in E to H = 22 μm, and bars in I to N = 52 μm.

Figure 4.

Overexpression of PtrWND2B in poplar induces the expression of a set of downstream transcription factors. Leaves of the PtrWND2B overexpressor seedlings as shown in Fig. 3 were used for real-time quantitative PCR analysis of the expression of poplar transcription factors that are close homologs of Arabidopsis SND-regulated downstream transcription factors (marked at the left of each group of poplar genes). The expression level of each gene in the control is set to 1. Error bars represent the se of three biological replicates.

Figure 5.

Up-regulation of the expression of additional transcription factors by overexpression of PtrWND2B in poplar. Leaves of the PtrWND2B overexpressors were used for real-time quantitative PCR analysis of the expression of poplar transcription factors. The expression level of each gene in the control is set to 1. Error bars represent the se of three biological replicates.

Figure 6.

Activation of the promoters of the PtrWND2B-induced poplar transcription factors by PtrWND2B, PtrWND6B, and EgWND1. The promoter activation was analyzed by cotransfecting Arabidopsis leaf protoplasts with the GUS reporter (top left panel) and the effector (top right panel) constructs. The GUS reporter constructs consist of the GUS reporter gene driven by the 2-kb promoter sequences of poplar transcription factors (TFs). The GUS activity in the protoplasts transfected with the GUS reporter construct only (control) is set to 1. Error bars represent the se of three biological replicates. NosT, Nopaline synthase terminator.

Figure 7.

Expression patterns of PtrWND-regulated transcription factors in the developing wood of wild-type poplar stems. Cross-sections of stems were hybridized with digoxigenin-labeled antisense (A–U) or sense (V) RNA probes, and the hybridization signals were detected with alkaline phosphatase-conjugated antibodies and are shown as purple color. Stem sections from three different plants were hybridized with each probe, and representative data are shown. The hybridization signals for each gene (labeled on each panel) were evident in vessels, xylary fibers, and ray parenchyma cells in the developing wood but not in the mature wood. Phloem fibers also showed positive hybridization signals. Stem sections hybridized with the control sense probes of each gene showed an absence of hybridization signals. One representative image for the sense probe of PtrNAC156 is shown (V). pf, Phloem fiber; sx, secondary xylem. Bar in A = 74 μm for A to V.

Figure 8.

Activation of the SNBE site from the Arabidopsis MYB46 promoter by WNDs. The ability of WNDs to activate the SNBE site was tested by cotransfecting Arabidopsis leaf protoplasts with the GUS reporter (top left panel) and the effector (top right panel) constructs. The GUS reporter constructs consist of the GUS reporter gene driven by three copies of the wild-type or mutated Arabidopsis MYB46 SNBE1 sequence. The bottom panel depicts the WND-activated expression of the GUS reporter gene driven by the wild-type or the mutated MYB46 SNBE1 sequence as indicated at left. The wild-type SNBE sequence was mutated by altering all the noncritical nucleotides (M1) or changing one or more critical nucleotides (M2–M5; Zhong et al., 2010c). The GUS activity in the protoplasts transfected with the GUS reporter construct only is set to 1. Error bars represent the se of three biological replicates.

Figure 9.

Activation of the SNBE sites from a number of Arabidopsis SND1 direct targets by WNDs. The ability of WNDs to activate the SNBE sites was tested by cotransfecting Arabidopsis leaf protoplasts with the GUS reporter and the effector constructs as described in Figure 8. The GUS reporter constructs consist of the GUS reporter gene driven by three copies of the SNBE sequences from the promoters of various SND1 direct targets. The GUS activity in the protoplasts transfected with the GUS reporter construct only (control) is set to 1. Error bars represent the se of three biological replicates.

Figure 10.

Direct binding of WNDs to the SNBE sites in the promoters of WND-regulated transcription factors. Purified recombinant WNDs fused with MBP was incubated with the biotin-labeled PtrMYB3 promoter fragment (−470 to −365 relative to the start codon) and subjected to EMSA. A, EMSA showing a band shift caused by binding of PtrWND2B (lane 3), PtrWND6B (lane 5), and EgWND1 (lane 7) to the labeled PtrMYB3 promoter fragment. No band shift was seen in the probe without the addition of proteins (lane 1) or with the addition of MBP (lane 2). Addition of the unlabeled PtrMYB3 promoter fragment (competitor) eliminated the band shift caused by WNDs. B, Representative SNBE sequences from the promoters of PtrWND-regulated transcription factors that are homologous to the Arabidopsis SND1 direct targets. These SNBE sequences were used for the EMSA competition analysis in C. The consensus nucleotides in the SNBE sequences are shaded. The mutated SNBE used in C is a mutated version of PtrMYB3 SNBE sequence with mutations of all nine consensus nucleotides. The number shown at the left of each sequence is the position of the first nucleotide relative to the start codon. C, SNBE sequences from the promoters of PtrWND-regulated transcription factors but not the mutated SNBE (mSNBE) effectively competed with the binding of PtrWND2B (top panel), PtrWND6B (middle panel), and EgWND1 (bottom panel) to the labeled PtrMYB3 promoter fragment. D, Transactivation test of the SNBE sequences by WNDs. The left panel depicts the GUS reporter and the effector constructs. The GUS reporter constructs consist of the GUS reporter gene driven by two copies of the SNBE sequences from the promoters of various PtrWND targets (B). The right panel shows the activation of the SNBE sequences by WNDs in Arabidopsis protoplasts cotransfected with the GUS reporter and the effector constructs. The GUS activity in the protoplasts transfected with the GUS reporter construct only (control) is set to 1. Error bars represent the se of three biological replicates.

Figure 11.

Overexpression of PtrWND2B induces the expression of genes involved in secondary wall biosynthesis, cell wall modification, and programmed cell death in poplar. A, Real-time quantitative PCR analysis showing the induction in the expression of genes involved in secondary wall biosynthesis, cell wall modification, and programmed cell death in the leaves of PtrWND2B overexpressors. The expression of each gene in the control is set to 1. Error bars represent the se of three biological replicates. B, Representative SNBE sequences from the promoters of several PtrWND-induced genes that are involved in secondary wall biosynthesis, cell wall modification, and programmed cell death. These SNBE sequences were used for the EMSA competition analysis in C. The consensus nucleotides in the SNBE sequences are shaded. The number shown at the left of each sequence is the position of the first nucleotide relative to the start codon. C, SNBE sequences from the promoters of several PtrWND-regulated genes efficiently competed with the binding of PtrWND2B (top panel), PtrWND6B (middle panel), and EgWND1 (bottom panel) to the labeled PtrMYB3 promoter fragment. D, Transactivation analysis of the SNBE sequences using the GUS reporter gene. The GUS reporter and the effector constructs are shown in the left panel. The GUS reporter constructs are made of the GUS reporter gene driven by two copies of the SNBE sequences from the promoters of various PtrWND targets (B). Activation of the SNBE sequences by WNDs was tested in Arabidopsis protoplasts by cotransfecting the GUS reporter and the effector constructs (right panel). The GUS activity in the protoplasts transfected with the GUS reporter construct only (control) is set to 1. Error bars represent the se of three biological replicates.

Figure 12.

Activation of the promoters of poplar secondary wall biosynthetic genes by PtrWND downstream transcription factors. Diagrams of the reporter and effector constructs used for the transactivation analysis are shown in the top left and top right panels, respectively. The effector constructs consist of the full-length cDNA of PtrWND downstream transcription factors (TFs) driven by the CaMV 35S promoter. The reporter constructs contain the GUS reporter gene driven by the 2-kb promoters of poplar secondary wall biosynthetic genes. Cotransfection of Arabidopsis leaf protoplasts with the GUS reporter and the effector constructs revealed the activation of the secondary wall gene promoters by several PtrWND downstream transcription factors. PtrWND2B/6B, EgWND1, PtrMYB3, and EgMYB2 were used as positive controls in the assay. The GUS activity in protoplasts transfected with the reporter construct alone was set to 1. Error bars represent the se of three biological replicates.

Figure 13.

Diagram of the transcriptional regulatory network controlling wood formation. The WNDs in poplar and Eucalyptus have been shown to function as the first-level master switches that directly activate a number of downstream transcription factors as well as many genes involved in secondary wall biosynthesis, cell wall modification, and programmed cell death. PtrMYB3 and PtrMYB20, direct targets of PtrWNDs, act as second-level master switches activating a secondary wall biosynthetic program (McCarthy et al., 2010). EgMYB2 from Eucalyptus and PtMYB4 from pine are functional orthologs of PtrMYB3/20. Among the PtrWND-regulated transcription factors, a number of them have been demonstrated to be able to activate the promoters of genes for all three secondary wall biosynthetic pathways, and a few others only act on the promoters of lignin biosynthetic genes in a transient transactivation system. In Arabidopsis, only the NAC and MYB master switches are known to activate all three secondary wall biosynthetic pathways. Note that for simplicity, many additional PtrWND-regulated transcription factors as listed in Figures 4 and 5 are not shown here. [See online article for color version of this figure.]