Functional characterization of poplar wood-associated NAC domain transcription factors

Abstract

Wood is the most abundant biomass produced by land plants. Dissection of the molecular mechanisms underlying the transcriptional regulation of wood formation is a fundamental issue in plant biology and has important implications in tree biotechnology. Although a number of transcription factors in tree species have been shown to be associated with wood formation and some of them are implicated in lignin biosynthesis, none of them have been demonstrated to be key regulators of the biosynthesis of all three major components of wood. In this report, we have identified a group of NAC domain transcription factors, PtrWNDs, that are preferentially expressed in developing wood of poplar (Populus trichocarpa). Expression of PtrWNDs in the Arabidopsis (Arabidopsis thaliana) snd1 nst1 double mutant effectively complemented the secondary wall defects in fibers, indicating that PtrWNDs are capable of activating the entire secondary wall biosynthetic program. Overexpression of PtrWND2B and PtrWND6B in Arabidopsis induced the expression of secondary wall-associated transcription factors and secondary wall biosynthetic genes and, concomitantly, the ectopic deposition of cellulose, xylan, and lignin. Furthermore, PtrWND2B and PtrWND6B were able to activate the promoter activities of a number of poplar wood-associated transcription factors and wood biosynthetic genes. Together, these results demonstrate that PtrWNDs are functional orthologs of SND1 and suggest that PtrWNDs together with their downstream transcription factors form a transcriptional network involved in the regulation of wood formation in poplar.

Figure 1.

Phylogenetic and expression analyses of PtrWNDs. A, Poplar NAC homologs of Arabidopsis SND1 were analyzed for their phylogenetic relationship with SND1, NSTs, and VNDs using the ClustalW program (Thompson et al., 1994), and the phylogenetic tree was displayed using the TREEVIEW program (Page, 1996). The 0.1 scale represents 10% change. Bootstrap values are shown in percentages at nodes. B, RT-PCR analysis of the expression levels of PtrWNDs in different poplar organs. Stem I and stem II are from elongating and nonelongating parts of stems, respectively. The expression of an actin gene is shown as an internal control.

Figure 2.

Cell type expression patterns of PtrWNDs in the developing wood of poplar stems. Cross sections of stems were hybridized with digoxigenin-labeled antisense (A–F) or sense (G–I) RNA probes, and the hybridization signals were detected with alkaline phosphatase-conjugated antibodies and are shown as purple color. Stem sections from at least five different plants were hybridized with each probe, and representative data are shown. A to F, The hybridization signals for PtrWND1 (A), PtrWND2 (B), PtrWND3 (C), PtrWND4 (D), PtrWND5 (E), and PtrWND6 (F) 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 for PtrWND2 (B), PtrWND4 (D), PtrWND5 (E), and PtrWND6 (F). G to I, Stem sections hybridized with the control sense probes of PtrWND2 (G), PtrWND4 (H), and PtrWND6 (I) showing the absence of hybridization signals. pf, Phloem fiber; rp, ray parenchyma; ve, vessel; xf, xylary fiber. Bar in A = 65 μm (for all panels).

Figure 3.

Subcellular localization and transcriptional activation analysis of PtrWNDs. A to D, An Arabidopsis protoplast (A; differential interference contrast image) coexpressing PtrWND1B-YFP (B) and SND1-CFP (for cyan fluorescent protein; C). Note that the signals of PtrWND1B-YFP are colocalized with those of SND1-CFP, which is known to be targeted to the nucleus (D). E to I, Arabidopsis protoplasts expressing PtrWND2B-YFP (E), PtrWND3B-YFP (F), PtrWND4B-YFP (G), PtrWND5B-YFP (H), and PtrWND6B-YFP (I), showing their nuclear localization. J, An Arabidopsis protoplast expressing YFP alone showing the fluorescent signals throughout the cytoplasm. K, Transcriptional activation analysis of PtrWNDs fused with the GAL4 DNA-binding domain (GAL4DB) in yeast showing their ability to activate the expression of the His-3 and β-Gal reporter genes.

Figure 4.

Complementation of the Arabidopsis snd1 nst1 double mutant by expression of PtrWNDs. A, Expression of PtrWNDs rescued the pendent stem phenotype of snd1 nst1. Expression of SND1 in snd1 nst1 was used as a positive control. B, Measurement of the breaking strength of stems of the snd1 nst1 double mutant, snd1 nst1 complemented with PtrWNDs or SND1, and the wild type. Each bar represents the breaking force of the inflorescence stem of individual plants. C, Restoration of lignified secondary walls in the interfascicular fibers of stems of the snd1 nst1 double mutant by expression of PtrWNDs. The bottom parts of 8-week-old stems were sectioned and stained for lignin with phloroglucinol-HCl. if, Interfascicular fiber; xy, xylem. Bar in C = 82 μm for C to J. [See online article for color version of this figure.]

Figure 5.

Induction of ectopic secondary wall deposition in the leaves by overexpression of PtrWND2B and PtrWND6B. The full-length cDNA of PtrWND2B or PtrWND6B driven by the CaMV 35S promoter was introduced into wild-type Arabidopsis. The leaves of 4-week-old wild-type (WT) and transgenic plants were examined for ectopic deposition of lignin, cellulose, and xylan. A, RT-PCR analysis showing the expression of PtrWND2B (PtrWND2B-OE) or PtrWND6B (PtrWND6B-OE) in the seedlings of three representative transgenic lines. The expression of the Arabidopsis EF1α gene was used as an internal control. B, Four-week-old seedlings of overexpressors of PtrWND2B (middle) and PtrWND6B (right) showing curly leaves compared with the wild type (left). C and D, Differential interference contrast (C) and lignin autofluorescence (D) images of a wild-type leaf showing the lignified secondary wall thickening in veins (ve). E to H, Differential interference contrast (E and G) and lignin autofluorescence (F and H) images of the leaf epidermis of PtrWND2B (E and F) and PtrWND6B (G and H) overexpressors showing ectopic deposition of secondary walls and lignin. I to K, Sections of leaves of PtrWND2B (I) and PtrWND6B (J) overexpressors showing ectopic deposition of secondary wall cellulose in the epidermis (arrows) compared with the wild type (K). L to N, Sections of leaves of PtrWND2B (L) and PtrWND6B (M) overexpressors showing ectopic deposition of xylan in the epidermis (arrows) compared with the wild type (N). Bar in C = 63 μm for C to H, and bar in I = 64 μm for I to N.

Figure 6.

Ectopic deposition of lignin, cellulose, and xylan in the epidermis of stems of the PtrWND2B and PtrWND6B overexpressors. Cross sections of stems of the wild type (A, D, and G) and overexpressors of PtrWND2B (B, E, and H) and PtrWND6B (C, F, and I) stained for lignin (A–C), cellulose (D–F), and xylan (G–I) are shown. Note the ectopic deposition of lignin, cellulose, and xylan in the epidermal cells (arrows) of PtrWND2B and PtrWND6B overexpressors compared with those of the wild type. co, Cortex; ep, epidermis; if, interfascicular fiber. Bar in A = 160 μm for all panels.

Figure 7.

Induction in the expression of secondary wall biosynthetic genes for cellulose (CesA4, CesA7, and CesA8), xylan (IRX8, IRX9, and FRA8), and lignin (4CL1 and CCoAOMT1) in the overexpressors of PtrWND2B and PtrWND6B. Seedlings of 3-week-old Arabidopsis plants were examined for the expression of secondary wall biosynthetic genes using quantitative PCR. The expression level of each gene in the wild type was set to 1. Error bars indicate se of three biological replicates.

Figure 8.

Induction in the expression of secondary wall-associated transcription factors by overexpression of PtrWND2B or PtrWND6B. Seedlings of 3-week-old Arabidopsis plants were examined for the expression of genes of interest using quantitative PCR. The expression level of each gene in the wild type was set to 1. Error bars represent se of three biological replicates.

Figure 9.

Direct activation of the MYB46, SND3, MYB103, and KNAT7 genes by PtrWND2B and PtrWND6B. A, Diagram of the constructs used for direct target analysis. PtrWND2 and PtrWND6 are translationally fused with the regulatory region of the HER, and its expression is driven by the CaMV 35S promoter. B to E, The expression of MYB46 (B), SND3 (C), MYB103 (D), and KNAT7 (E) was induced by estradiol treatment (left panels) of protoplasts expressing PtrWND2B-HER or PtrWND6B-HER, and the induction still occurred in the presence of the protein synthesis inhibitor cycloheximide (CHX; right panels). The Arabidopsis protoplasts were transfected with PtrWND2B-HER or PtrWND6B-HER and then treated with estradiol or cycloheximide plus estradiol. The expression of each gene was detected using quantitative PCR. The expression level of each gene in the mock-treated (control) or cycloheximide-treated protoplasts was set to 1. Error bars denote se of three biological replicates.

Figure 10.

Activation of the promoters of poplar wood-associated transcription factors by PtrWND2B and PtrWND6B. A and B, Diagrams of the effector and reporter constructs used for the activation analysis. The effector constructs consist of the full-length cDNA of PtrWND2B or PtrWND6B driven by the CaMV 35S promoter (A). The reporter constructs contain the GUS reporter gene driven by the 2-kb promoters of the poplar transcription factors (B). NosT, Nopaline synthase terminator. C, A list of Arabidopsis secondary wall-associated transcription factors regulated by SND1 and their corresponding putative orthologs in poplar. D, Transactivation analysis showing the PtrWND2B- or PtrWND6B-activated expression of the GUS reporter gene driven by the promoters of poplar transcription factors. The effector and reporter constructs were cotransfected into Arabidopsis protoplasts, which were further subjected to GUS reporter gene expression analysis. GUS activity in protoplasts transfected with the reporter construct alone was used as a control and was set to 1. Error bars represent se of three biological replicates.

Figure 11.

Activation of the promoters of poplar secondary wall biosynthetic genes by PtrWND2B and PtrWND6B. A and B, Diagrams of the effector and reporter constructs used for the expression analysis. The effector constructs consist of the full-length cDNA of PtrWND2B or PtrWND6B driven by the CaMV 35S promoter (A). The reporter constructs contain the GUS reporter gene driven by the 2-kb promoters of poplar secondary wall biosynthetic genes (B). NosT, Nopaline synthase terminator. C, Transactivation analysis showing the PtrWND2B- or PtrWND6B-activated expression of the GUS reporter gene driven by the promoter of poplar PtrCesA8, PtrGT43B, or PtrCCoAOMT1. GUS activity in protoplasts transfected with the reporter construct alone was used as a control and was set to 1. Error bars represent se of three biological replicates.