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Plant Physiol. Feb 2010; 152(2): 1044–1055.
PMCID: PMC2815876

Functional Characterization of Poplar Wood-Associated NAC Domain Transcription Factors1,[C][OA]

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.

Wood (also called secondary xylem) is a complex tissue made of longitudinally and transversely arranged cell systems. The longitudinal system consists mainly of tracheids in gymnosperms and vessels and fibers in angiosperms. These cells are responsible for the conduction of water and minerals and the mechanical support of the plant body. The transverse system is composed of ray parenchyma cells that function in short-distance transverse conduction and nutrient storage. The longitudinal and transverse systems are derived from fusiform initials and ray initials, respectively, in the vascular cambium (Raven et al., 1999). Because of the complexity of wood structure, it is conceivable that the molecular mechanisms governing the differentiation of wood tissues are complicated. There has been a tremendous interest in understanding the molecular mechanisms underlying wood formation in the hope of genetically engineering the quality and quantity of wood for its better utilization. Being the most abundant biomass produced by land plants, wood is considered to be one of the most environmentally cost-effective and renewable sources of bioenergy (Han et al., 2007) and has been widely used for timber, pulping, paper making, and many other commercial applications.

Wood formation involves sequential developmental events, including differentiation of xylem mother cells from the vascular cambium, division of xylem mother cells, cell expansion, secondary wall thickening, programmed cell death, and finally, formation of heart wood (Plomion et al., 2001). To genetically modify wood property, it is essential to uncover the regulatory genes that control the various developmental events of wood formation and to identify the biosynthetic genes that are responsible for the biosynthesis of wood components, including cellulose, hemicellulose, and lignin. In the last several decades, molecular and genomic studies have revealed a number of wood-associated biosynthetic genes involved in the biosynthesis of cellulose, xylan, glucomannan, and lignin (Boerjan et al., 2003; Mellerowicz and Sundberg, 2008). In addition, a number of transcription factor genes have been demonstrated to be associated with wood formation (Patzlaff et al., 2003a, 2003b; Karpinska et al., 2004; Schrader et al., 2004; Goicoechea et al., 2005; Prassinos et al., 2005; Andersson-Gunneras et al., 2006; Bedon et al., 2007; Legay et al., 2007; Bomal et al., 2008; Wilkins et al., 2009). Among them, the best-studied ones are several MYB transcription factors from pine (Pinus spp.) and Eucalyptus. Overexpression of the pine PtMYB4 and the Eucalyptus EgMYB2 in tobacco (Nicotiana tabacum) results in ectopic deposition of lignin and increased thickness of secondary walls in xylem, respectively (Patzlaff et al., 2003a; Goicoechea et al., 2005). Two other pine MYBs, PtMYB1 and PtMYB8, have been demonstrated to induce ectopic lignin deposition and to alter the phenylpropanoid metabolism when overexpressed in spruce (Picea abies; Bomal et al., 2008). It has been proposed that these pine and Eucalyptus MYB genes are involved in the regulation of lignin biosynthesis during wood formation (Wilkins et al., 2009). Currently, no wood-associated transcription factors have been reported to be master switches activating the entire biosynthetic program for all wood components in a tree species.

Even though little is known about the transcriptional regulation of wood formation in tree species, a transcriptional network regulating secondary wall biosynthesis has been uncovered in Arabidopsis (Arabidopsis thaliana; Zhong and Ye, 2007). It has been demonstrated that a group of NAC domain transcription factors, including NST1, NST2, SND1 (also called NST3/ANAC012), VND6, and VND7, function as master switches activating the secondary wall biosynthetic program in fibers, vessels, or endothecium of anthers (Kubo et al., 2005; Mitsuda et al., 2005, 2007; Zhong et al., 2006, 2007b, 2008; Yamaguchi et al., 2008). These NACs have been shown to regulate the expression of a battery of secondary wall-associated transcription factors. Among them, MYB46, MYB83, SND3, MYB103, and KNAT7 were proven to be their direct targets and MYB58, MYB63, and MYB85 were found to specifically regulate lignin biosynthesis (Zhong et al., 2007a, 2008; McCarthy et al., 2009; Zhou et al., 2009). These studies have demonstrated that a complex transcriptional network comprising a cascade of transcription factors is involved in the regulation of secondary wall biosynthesis in Arabidopsis.

Despite the fact that Arabidopsis plants undergo a certain degree of secondary growth as in tree species (Chaffey et al., 2002; Ye et al., 2002), the structure and composition of wood in tree species exhibit some distinct features from those of the xylem tissues in Arabidopsis. It is conceivable that in addition to the common mechanisms regulating vascular tissue formation shared by all vascular plants, tree species might have evolved unique regulatory mechanisms controlling wood formation (Nieminen et al., 2004). Therefore, although knowledge gained from the study of secondary wall biosynthesis in the herbaceous Arabidopsis could be applied to further our understanding of wood formation, it is still essential to identify and functionally characterize wood-associated regulatory genes from tree species (Wilkins et al., 2009). In this report, we demonstrate that a group of poplar (Populus trichocarpa) NAC domain transcription factors (PtrWNDs) are functional orthologs of the Arabidopsis SND1. We show that PtrWNDs are expressed not only in developing fibers and vessels but also in ray parenchyma cells. Overexpression of two of them, PtrWND2B and PtrWND6B, leads to ectopic deposition of secondary walls in Arabidopsis. Furthermore, we reveal that PtrWND2B and PtrWND6B are able to activate the promoter activities of a number of poplar wood-associated transcription factors and wood biosynthetic genes. Our findings suggest that the wood-associated PtrWNDs together with their downstream transcription factors are involved in the regulation of wood formation in poplar.

RESULTS

Wood-Associated Expression of a Group of NAC Domain Transcriptional Activators

As a first step toward dissecting the molecular mechanisms underlying the transcriptional regulation of wood formation, we searched the poplar genome for NAC domain transcription factors that show high sequence similarity to the Arabidopsis SND1. A total of 16 poplar SND1 homologs were identified, and they phylogenetically correspond to members of a set of Arabidopsis NAC genes that are implicated in transcriptional regulation of secondary wall biosynthesis or xylem differentiation (Fig. 1A; Kubo et al., 2005; Mitsuda et al., 2005, 2007; Zhong et al., 2006, 2007b). These poplar NACs apparently fall into eight pairs; the members of each pair share at least 88% similarity in their amino acid level and thus likely originated from genome duplication (Tuskan et al., 2006). Therefore, only one gene representing each pair was used for functional characterization as described below. The expression of PtrNAC054, PtrNAC058, PtrNAC064, and PtrNAC066 was not detected in woody stems; therefore, they were not characterized in this study. The rest of the poplar NAC genes were highly expressed in stem tissues in which wood formation occurs (Fig. 1B); thus, they were named PtrWNDs (for wood-associated NAC domain transcription factors).

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 ...

We next used the in situ hybridization approach to examine the cell type-level expression patterns of PtrWNDs in the developing wood of poplar. Wood cells are composed of longitudinally oriented vessels and fibers that undergo secondary wall thickening and transversely oriented ray parenchyma cells, a structural organization distinct from that of the herbaceous Arabidopsis. It was found that all PtrWND genes were expressed prominently in developing vessels and fibers in the developing wood (Fig. 2, A–F), indicating their close association with cells undergoing secondary wall biosynthesis. Strong expression signals were also observed in phloem fibers for PtrWND2, PtrWND4, PtrWND5, and PtrWND6. Interestingly, the PtrWND transcripts were also evident in ray parenchyma cells, a cell type that is involved in transverse conduction and storage. It should be noted that each probe recognizes the transcripts of both genes in the pair due to their high sequence identity, but it does not hybridize with those of other PtrWNDs. No hybridization signals were observed in wood cells in the control sections hybridized with the sense probes (Fig. 2, G–I). These results demonstrate that PtrWNDs are expressed in all cell types of developing wood.

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 ...

Sequence analysis showed that the PtrWND proteins possess the typical domain organization of known plant NAC transcription factors, including the NAC domain located at the N terminus and the putative activation domain at the C terminus (Olsen et al., 2005). Subcellular localization using the fluorescent protein-tagging approach demonstrated that while yellow fluorescent protein (YFP) alone exhibited a dispersed cytoplasmic distribution (Fig. 3J), YFP-tagged PtrWNDs were targeted to the nucleus (Fig. 3, A–I), consistent with their functions as transcription factors. To further ascertain whether they were able to activate transcription, we fused PtrWNDs with the GAL4 DNA-binding domain and tested their ability to activate the reporter gene expression in yeast. It was found that PtrWNDs were able to activate the expression of the His-3 and β-Gal reporter genes (Fig. 3 K), indicating that they are transcriptional activators.

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 ...

Expression of PtrWNDs Complements the Secondary Wall Defects in the Arabidopsis snd1 nst1 Double Mutant

The finding that all the PtrWND genes are expressed in developing poplar wood indicates that they may play redundant roles in the regulation of wood formation. To circumvent the difficulty in the functional study of redundant genes in trees, we resorted to the Arabidopsis snd1 nst1 double mutant to investigate whether PtrWNDs are functional orthologs of Arabidopsis SND1 involved in the regulation of secondary wall biosynthesis. The Arabidopsis mutant complementation approach has been successfully applied to analyze the functions of poplar genes involved in the biosynthesis of wood components (Zhou et al., 2006, 2007). To do this, the full-length cDNAs of PtrWNDs driven by the Arabidopsis SND1 promoter were expressed in the Arabidopsis snd1 nst1 double T-DNA knockout mutant. Simultaneous mutations of SND1 and NST1 in Arabidopsis were previously shown to cause a loss of secondary walls in fibers and, concomitantly, a reduction in stem strength and a pendent stem phenotype (Mitsuda et al., 2007; Zhong et al., 2007b). Expression of PtrWNDs in the snd1 nst1 double mutant rescued the pendent inflorescence stem phenotype and significantly restored the stem strength (Fig. 4, A and B). In particular, PtrWND2B, PtrWND5B, and PtrWND6B functioned as effectively as SND1 itself, since the break strength of the stems of their complemented mutant plants was comparable to that of the SND1 complemented plants and the wild type. The recovery of stem strength in the complemented snd1 nst1 plants was accompanied by a restoration in the deposition of secondary walls in interfascicular fibers (Fig. 4, C–J). These results demonstrate that PtrWNDs are able to complement the secondary wall defects caused by the snd1 nst1 double mutation and suggest that PtrWNDs are functional orthologs of SND1 and NST1.

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 ...

Overexpression of PtrWND2B and PtrWND6B Induces Ectopic Deposition of Secondary Walls in Arabidopsis

To further investigate the roles of PtrWNDs in the transcriptional regulation of secondary wall biosynthesis, we expressed PtrWNDs driven by the cauliflower mosaic virus (CaMV) 35S promoter in wild-type Arabidopsis. At least 124 transgenic plants expressing each of the PtrWNDs were generated and examined for their morphological and ectopic secondary wall deposition phenotypes. It was found that about 20% of the seedlings with overexpression of PtrWND2B or PtrWND6B had severely curled leaves (Fig. 5, A and B), a phenotype similar to that exhibited by Arabidopsis SND1 or NST1 overexpressors (Mitsuda et al., 2005; Zhong et al., 2006). The transgenic plants overexpressing other PtrWNDs did not show a visible curly leaf phenotype. To find out whether the curly leaf phenotype was due to ectopic deposition of secondary walls, we examined the presence of the three major secondary wall components, cellulose, xylan, and lignin, in leaf parenchyma cells. Although lignin was only present in the veins of wild-type leaves (Fig. 5, C and D), ectopic deposition of lignin was evident in the leaf epidermis of PtrWND2B and PtrWND6B overexpressors (Fig. 5, E–H). It was noted that the ectopic lignin deposition was associated with thickened walls with helical and reticulated patterns (Fig. 5, E–H). In addition to lignin, ectopic deposition of secondary wall cellulose and xylan was also detected in the leaf epidermis of PtrWND2B and PtrWND6B overexpressors compared with that of the wild type (Fig. 5, I–N). Similarly, the ectopic deposition of lignin, cellulose, and xylan was also observed in the epidermis of the stems of PtrWND2B and PtrWND6B overexpressors (Fig. 6). The phenomenon that the ectopic deposition of secondary walls only occurred in some parenchyma cells in the overexpressors is also common in plants overexpressing SND1 or NST1 (Mitsuda et al., 2005; Zhong et al., 2006), which is probably due to the differential competence of cells to become sclerified.

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 ...
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 ...

The finding that overexpression of PtrWND2B or PtrWND6B induces ectopic deposition of secondary walls prompted us to analyze the expression levels of secondary wall biosynthetic genes. Real-time quantitative PCR analysis demonstrated that the secondary wall biosynthetic genes, including cellulose synthases (CesA4, CesA7, and CesA8; Taylor et al., 2004), xylan biosynthetic genes (FRA8, IRX8, and IRX9; Pena et al., 2007), and lignin biosynthetic genes (4CL1 and CCoAOMT1; Raes et al., 2003), were all induced in the overexpressors of PtrWND2B and PtrWND6B compared with the wild type (Fig. 7). These results demonstrate that PtrWND2B and PtrWND6B are able to activate the expression of secondary wall biosynthetic genes and concomitantly result in ectopic deposition of secondary walls.

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 ...

Up-Regulation of the Expression of Arabidopsis Secondary Wall-Associated Transcription Factors by PtrWND2B and PtrWND6B

We next examined whether overexpression of PtrWND2B and PtrWND6B in Arabidopsis induced the expression of secondary wall-associated transcription factors that are members of the SND1-mediated transcriptional network (Zhong et al., 2008). Real-time quantitative PCR analysis showed that the expression of 15 secondary wall-associated transcription factors was up-regulated in the overexpressors of PtrWND2B and PtrWND6B (Fig. 8), indicating that PtrWND2B and PtrWND6B are able to activate the expression of downstream transcription factors implicated in secondary wall biosynthesis. We further investigated whether PtrWND2B and PtrWND6B could directly activate the expression of the four known direct targets of SND1, including MYB46, SND3, MYB103, and KNAT7 (Zhong et al., 2008). PtrWND2B or PtrWND6B fused with the regulatory domain of the human estrogen receptor (HER) was expressed under the control of the CaMV 35S promoter in Arabidopsis protoplasts (Fig. 9A). Real-time quantitative PCR analysis revealed that the expression of MYB46, SND3, MYB103, and KNAT7 was induced by estradiol activation of PtrWND2B-HER or PtrWND6B-HER, and the induction still occurred in the presence of the protein synthesis inhibitor cycloheximide (Fig. 9, B–E) and thus is direct. The lower induction level in the expression of these genes in the presence of cycloheximide is most likely due to the inhibition of the overall protein synthesis by cycloheximide. These results provide additional lines of evidence suggesting that, like SND1, PtrWND2B and PtrWND6B function as master switches activating the transcriptional network regulating secondary wall biosynthesis.

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 ...
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 ...

Activation of the Promoters of Poplar Wood-Associated Transcription Factors and Wood Biosynthetic Genes by PtrWND2B and PtrWND6B

The finding that PtrWND2B and PtrWND6B activate the expression of a number of secondary wall-associated transcription factors and secondary wall biosynthetic genes in Arabidopsis prompted us to investigate whether they also regulate the expression of poplar wood-associated transcription factors and wood biosynthetic genes. A search of the poplar genome revealed the existence of close homologs corresponding to the 15 known SND1 downstream transcription factors (Fig. 10C; Tuskan et al., 2006). Most of these poplar transcription factors are highly expressed in developing wood according to the transcriptome data from the Poplar eFP Browser (http://www.bar.utoronto.ca/efppop/cgi-bin/efpWeb.cgi; Wilkins et al., 2009), indicating that they might be involved in wood formation. One gene from each group of the poplar transcription factors was chosen for the examination of transactivation of their promoters by PtrWND2B and PtrWND6B. The 2-kb promoters of these genes were linked with the GUS reporter gene to create the reporter constructs, and the full-length cDNAs of PtrWND2B and PtrWND6B were ligated downstream of the CaMV 35S promoter to generate the effector constructs (Fig. 10, A and B). The reporter and effector constructs were cotransfected into Arabidopsis protoplasts, and subsequent assay of the GUS activity in the transfected protoplasts demonstrated that PtrWND2B and PtrWND6B effectively activated the promoters of most of these wood-associated transcription factors (Fig. 10D). The only exceptions are the promoter of PtrMYB018, which was not activated by PtrWND2B and PtrWND6B, and those of PtrKNAT7 and PtrMYB128, which were not activated by PtrWND6B.

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 ...

We next examined whether the promoters of poplar wood biosynthetic genes could be activated by PtrWND2B and PtrWND6B. The promoters of one representative biosynthetic gene for each wood component were chosen for transactivation analysis (Fig. 11A). These wood biosynthetic genes are PtrCesA8 (also called PtiCesA8-A), involved in cellulose biosynthesis (Suzuki et al., 2006), PtrGT43B, involved in xylan biosynthesis (Zhou et al., 2007), and PtrCCoAOMT1, involved in lignin biosynthesis (Boerjan et al., 2003). Protoplasts cotransfected with the effector and reporter constructs (Fig. 11A) showed high GUS activity, indicating that PtrWND2B and PtrWND6B are capable of activating the expression of poplar wood biosynthetic genes. Together, these results suggest that PtrWND2 and PtrWND6 are involved in regulation of the expression of a number of wood-associated transcription factors and wood biosynthetic genes in poplar.

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 ...

DISCUSSION

Previous molecular and genomic studies on wood formation have placed major efforts on the identification of the biosynthetic genes of wood components, which is the first important step toward our understanding of wood formation and our attempts to genetically modify wood quantity and quality (Mellerowicz and Sundberg, 2008). To modify wood property, one common strategy is to either down- or up-regulate the biosynthetic genes of wood components. It is relatively straightforward to reduce the amount of wood components by simply down-regulating one or a few biosynthetic genes. However, it might be much more complicated to increase the amount of wood components, because wood biosynthesis requires the coordinated activation of all biosynthetic pathways involved in wood formation. To further enrich our tools for genetic modification of wood, it is imperative to uncover master regulatory switches, such as transcription factors, that are able to activate the entire biosynthetic program for wood components. Our finding that the poplar NAC domain transcription factors, PtrWNDs, are SND1 functional orthologs capable of activating the expression of poplar wood-associated transcription factors and wood biosynthetic genes marks an important step toward this direction.

We have demonstrated that all of the poplar PtrWNDs examined are expressed in developing wood and are capable of complementing the secondary wall defects in the Arabidopsis snd1 nst1 mutant, indicating that they are functional orthologs of Arabidopsis SND1. Although the Arabidopsis secondary wall NACs exhibit fiber- or vessel-specific expression (Kubo et al., 2005; Zhong et al., 2006, 2008; Mitsuda et al., 2007; Yamaguchi et al., 2008), PtrWNDs are expressed in both vessels and fibers in developing wood. This finding suggests that multiple functionally redundant PtrWNDs are involved in the regulation of wood formation. Considering the facts that herbaceous Arabidopsis and woody poplar shared their last common ancestor over 100 million years ago (Tuskan et al., 2006) and wood formation is crucial for the survival of tree species, it is conceivable that poplar trees evolved to have more complexity of functionally redundant regulatory switches to ensure the process of wood formation compared with the herbaceous Arabidopsis plants with only a limited secondary growth. It is noteworthy that PtrWNDs are also expressed in ray parenchyma cells in developing wood. Because the biosynthetic genes for wood components, including cellulose, xylan, and lignin, have been shown to be expressed in ray parenchyma cells (Feuillet et al., 1995; Kalluri and Joshi, 2004; Zhou et al., 2006), it is likely that PtrWNDs are also involved in activation of the expression of wood biosynthetic genes in these cells.

We have further demonstrated that PtrWND2B and PtrWND6B are able to induce ectopic deposition of secondary walls when overexpressed in Arabidopsis, suggesting that they are master switches activating secondary wall biosynthesis during wood formation in poplar. It is interesting that overexpression of other PtrWNDs did not cause ectopic deposition of secondary walls, although they were able to complement the secondary wall defects in the snd1 nst1 mutant. One plausible explanation for this observation is that PtrWNDs have different activation strength and only PtrWND2B and PtrWND6B are active enough to result in ectopic deposition of secondary walls in parenchyma cells when overexpressed. In cells that are programmed to be sclerified, there exist additional transcription factors that might act cooperatively with the secondary wall NACs to activate secondary wall biosynthesis. Therefore, the PtrWNDs with low activation strength could restore secondary wall biosynthesis in the fibers of snd1 nst1. The different activation strength of PtrWNDs might have important implications in the regulation of secondary wall biosynthesis. Because NAC proteins may form homodimers or heterodimers for their transcriptional activities (Olsen et al., 2005), it is likely that a different combination of heterodimers of PtrWNDs may be involved in fine-tuning the regulation of downstream genes during wood formation.

In Arabidopsis, it has been shown that a transcriptional regulatory network encompassing the NAC master switches and their downstream targets is involved in the activation of the secondary wall biosynthetic program (Zhong and Ye, 2007). Although a number of transcription factors have been identified to be associated with wood formation in tree species and some of them have been implicated in the regulation of lignin biosynthesis (Karpinska et al., 2004; Patzlaff et al., 2003a, 2003b; Schrader et al., 2004; Goicoechea et al., 2005; Prassinos et al., 2005; Andersson-Gunneras et al., 2006; Bedon et al., 2007; Legay et al., 2007; Bomal et al., 2008; Wilkins et al., 2009), it is not clear whether a transcriptional network is involved in the regulation of wood formation in tree species. Our finding that PtrWND2B and PtrWND6B are able to activate the promoters of a number of poplar wood-associated transcription factors indicates that PtrWNDs regulate their expression. Because these wood-associated transcription factors are close homologs of members of the SND1-mediated transcription network, it is reasonable to propose that PtrWNDs together with their downstream transcription factors form a transcriptional network activating the secondary wall biosynthetic program during wood formation in poplar.

It has recently been reported that burning biomass in power plants to produce electricity for battery-driven vehicles captures more biomass energy than converting it to biofuels (Campbell et al., 2009), and it was suggested that future research should focus on increasing the yield and energy density of biomass in biomass crop plants (Ohlrogge et al., 2009). Our identification of wood-associated PtrWNDs as key regulators of secondary wall biosynthesis will potentially provide unprecedented tools to increase biomass production in biomass crop plants. Further studies on the transcriptional regulatory network mediated by PtrWNDs will undoubtedly advance our understanding of the transcriptional regulation of wood formation.

MATERIALS AND METHODS

Gene Expression Analysis

Total RNA was isolated from poplar (Populus trichocarpa) plants with a Qiagen RNA isolation kit. Plants were grown in a greenhouse under 14-h-light/10-h-dark cycles with supplemental light. The expanding leaves and petioles were from the top four expanding leaves, elongating stems included the first and second internodes, and nonelongating stems included the fourth and fifth internodes from the top of the stems (1 m height). Samples from at least 10 stems were pooled for analysis. First-strand cDNAs were synthesized from the total RNA treated with DNase I and then used as a template for PCR analysis. The primers used are as follows: PtrWND1B (5′-TTGGTTCAGGAAATGATGGA-3′ and 5′-TTATACCGATAAGTGGCATAATGG-3′), PtrWND2B (5′-CTTGCAATGAGGGCTCTCTAGATC-3′ and 5′-CTATACACTAGTGTTTGGCAAGTG-3′), PtrWND3B (5′-TAGATCTGCAGTACCAAATCCCTC-3′ and 5′-TCATTTCCATAGATCAATTTGACAACTG-3′), PtrWND4B (5′-CTCAACCTTATGCATCATACCTTCATC-3′ and 5′-TCACTTCCACAGATCAATTTGACAAC-3′), PtrWND5B (5′-GCTCAGTGGTGGATCCTATTG-3′ and 5′-TCATTTTTCAAATATGCATATTCCAATATC-3′), and PtrWND6B (5′-GCTTGGAGTCATGCTTATTATCTC-3′ and 5′-TAAGTCAGGAAAGCAGTCAAGGA-3′). The expression level of an actin gene served as an internal control to determine the reverse transcription (RT)-PCR amplification efficiency among different samples. Various PCR cycles were performed to determine the logarithmic phase of amplifications for the samples. RT-PCR was repeated three times, and similar results were obtained.

Real-time quantitative PCR was performed with the QuantiTect SYBR Green PCR kit (Clontech) using first-strand cDNAs as templates. The relative expression level was calculated by normalizing the PCR threshold cycle number of each gene with that of the EF1α reference gene. The data were averages of three biological replicates from three different overexpresser plants or three independent protoplast transfection experiments.

In Situ Hybridization

The fourth and fifth internodes of the stems (1 m height) of poplar plants growing in a greenhouse were fixed in 2.5% formaldehyde and 0.5% glutaraldehyde, embedded in paraffin, and sectioned (12 μm thick) for in situ mRNA localization according to McAbee et al. (2005) and Zhou et al. (2007). The 500-bp 3′ sequences of PtrWND cDNAs without the NAC domain sequences were used for synthesis of digoxigenin-labeled antisense and sense RNA probes with the DIG RNA Labeling mix (Roche). Stem sections were hybridized with the antisense and sense probes, and the hybridization signals were detected by incubating with alkaline phosphatase-conjugated antibodies against digoxigenin and subsequent color development with alkaline phosphatase substrates.

Subcellular Localization and Transcriptional Activation

The subcellular localization of PtrWNDs was performed by transfecting YFP-tagged PtrWNDs into Arabidopsis (Arabidopsis thaliana) leaf protoplasts (Sheen, 2001). The full-length cDNAs of PtrWNDs were fused in frame with the YFP cDNA and ligated between the CaMV 35S promoter and the nopaline synthase terminator. The fluorescence signals in transfected protoplasts were examined using a TCs SP2 spectral confocal microscope (Leica Microsystems). Images were saved and processed with Adobe Photoshop version 7.0 (Adobe Systems).

The transcriptional activation activity of PtrWNDs was tested by transforming the pAS2-1 construct containing a fusion of PtrWNDs and the GAL4 DNA-binding domain into the yeast strain CG-1945. The yeast strain contains the His-3 and LacZ reporter genes. The transformed yeast cells were grown on synthetic defined plates with or without His and assayed for β-galactosidase activity.

Complementation of the snd1 nst1 Mutant

The full-length cDNAs of PtrWNDs driven by the 3-kb SND1 promoter were cloned into the pGPTV binary vector. The constructs were introduced into the Arabidopsis snd1 nst1 double mutant (Zhong et al., 2007b) by Agrobacterium tumefaciens-mediated transformation (Bechtold and Bouchez, 1994). Transgenic plants were selected on hygromycin, and the first generation of transgenic plants was used for breaking strength and anatomical analyses. Basal parts of the main inflorescence of 8-week-old plants growing in a greenhouse under 14-h-light/10-h-dark cycles were measured for the breaking force using a digital force/length tester (Zhong et al., 1997). The breaking force was calculated as the force needed to break apart a stem segment. The basal parts of stems from eight independent transgenic plants were cut into 50-μm-thick sections and then stained for lignin with phloroglucinol-HCl.

Overexpression

The overexpression constructs were created by cloning the full-length cDNAs of PtrWNDs downstream of the CaMV 35S promoter in pBI121. The constructs were introduced into wild-type Arabidopsis plants (ecotype Columbia) by Agrobacterium-mediated transformation. Transgenic plants were selected on kanamycin, and the first generation of transgenic plants (at least 124 plants for each construct) was examined for their phenotypes. At least 10 independent transgenic plants exhibiting severe phenotypes were selected for phenotypic characterization, and representative results are presented. Leaf and stem tissues were fixed and sectioned for cellulose, xylan, and lignin staining. Lignin was examined by staining sections with phloroglucinol-HCl or visualized using a UV fluorescence microscope (Zhong et al., 2006). Secondary wall cellulose staining was done by incubating 1-μm-thick sections with 0.01% Calcofluor White (Hughes and McCully, 1975). Xylan was detected using the monoclonal LM10 antibody against xylan and fluorescein isothiocyanate-conjugated goat anti-rat secondary antibodies according to McCartney et al. (2005).

Direct Target Analysis

The full-length cDNAs of PtrWND2 and PtrWND6 were fused with the regulatory region of the HER (Zuo et al., 2000) in pBI221 to create the estrogen-inducible expression constructs. The constructs were transfected into Arabidopsis leaf protoplasts (Sheen, 2001) and then treated with 2 μm estrogen for 6 h before being harvested for RNA isolation. New protein synthesis in the transfected protoplasts was inhibited by addition of the protein synthesis inhibitor cycloheximide (2 μm). Under this condition, new protein synthesis was completely inhibited as tested by GUS reporter gene analysis (Zhong et al., 2008). Gene expression was examined with quantitative PCR analysis, and the expression level in the control was set to 1. The data are averages of three biological replicates. The primers of Arabidopsis genes analyzed were described previously (Zhong et al., 2006; McCarthy et al., 2009; Zhou et al., 2009).

Transactivation Analysis

For testing the ability of PtrWNDs to activate the poplar gene promoters, the reporter construct containing the GUS reporter gene driven by a 2-kb promoter of the poplar gene of interest and the effector construct containing PtrWNDs driven by the CaMV 35S promoter were cotransfected into Arabidopsis leaf protoplasts. Another construct containing the firefly luciferase gene driven by the CaMV 35S promoter was also included in the transfection for the determination of transfection efficiency. After 20 h of incubation, protoplasts were lysed and the supernatants were subjected to assay of the GUS and luciferase activities (Gampala et al., 2001). The GUS activity was normalized against the luciferase activity in each transfection, and the data are averages of three biological replicates. The primers used for PCR amplification of the poplar gene promoters are as follows: PtrNAC156 (5′-ACTGACTTAAGATTTGAATGCTGA-3′ and 5′-CTTAGGTTTCCTCTTATGCCCACA-3′), PtrNAC157 (5′-CGCATAAGCTTCTGTTGCGTAGCT-3′ and 5′-TATCTTATTCCCATCCCACAAAG-3′), PtrKNAT7 (5′-TCAACTCCAGCTCACTCCTGTGAC-3′ and 5′-GTAAATATTAGCACTAAAGATTCA-3′), PtrMYB018 (5′-AGGAATTACCACAATTGAGCGATG-3′ and 5′-TTTCACAAGATCACAAACACCAA-3′), PtrMYB020 (5′-GGTTTGGACAATGACTAATTTGAA-3′ and 5′-GATAGTATACTCTGATCAGCCAGC-3′), PtrMYB028 (5′-ACATTATGCAGTGTTAGGGTGAG-3′ and 5′-GATCTCTCTCAAATTTGCTAGCAA-3′), PtrMYB075 (5′-TTGGAGCTAAACCATGGGAGC-3′ and 5′-CACTATAGTTCAAGTCACTAGTTG-3′), PtrMYB090 (5′-AACCTATCGAACCCATAAGCTAGG-3′ and 5′-CAATCAAAGAATCCAATCCCAAG-3′), PtrMYB128 (5′-ATTTTCATGAGGTCTCTGGATGTC-3′ and 5′-CTATGATATATCTGATCTTATTG-3′), PtrMYB158 (5′-GATTGTTCAGAGGGACTCTCAAC-3′ and 5′-TTACCCCTATCTATCTAGCTTTGA-3′), PtrCesA8 (5′-GAATCCTGCATTTGTTCATGCA-3′ and 5′-GCTGATTACCACTTCACCGGCT-3′), PtrGT43B (5′-ATATTCTCAACACTATAGTCATTG-3′ and 5′-GCTAAACCCCTCAAAAACGTG-3′), and PtrCCoAOMT1 (5′-TGTAGCTTACTTAGTTAGCATACG-3′ and 5′-GTGTATATCTTCTAATTAAACTAC-3′).

Statistical Analysis

The experimental data of the quantitative PCR analysis and GUS activity assay were subjected to statistical analysis using the Student's t test program (http://www.graphpad.com/quickcalcs/ttest1.cfm), and the quantitative difference between the two groups of data for comparison in each experiment was found to be statistically significant (P < 0.001).

The GenBank accession numbers and gene models for the poplar NAC genes are as follows: PtrWND1A (NAC068; XM_002317023; estExt_Genewise1_v1.C_LG_XI0943), PtrWND1B (NAC063; XM_002300464; estExt_Genewise1_v1.C_LG_I5469), PtrWND2A (NAC065; XM_002320861; eugene3.00140502), PtrWND2B (NAC061; XM_002302636; fgenesh1_pg.C_LG_II001661), PtrWND3A (NAC050; XM_002322362; fgenesh1_pm.C_LG_XV000316), PtrWND3B (NAC037; XM_002318252; eugene3.00121251), PtrWND4A (NAC038; XM_002329829; fgenesh1_pg.C_scaffold_127000046), PtrWND4B (NAC046; XM_002304392; fgenesh1_pg.C_LG_III000910), PtrWND5A (NAC025; XM_002310261; grail3.0019029001), PtrWND5B (NAC039; XM_002327730; grail3.0057010601), PtrWND6A (NAC055; XM_002327206; eugene3.00410078), PtrWND6B (NAC060; XM_002325955; eugene3.00190755), PtrNAC054 (XM_002316127; eugene3.00101697), PtrNAC058 (XM_002311240; eugene3.00080734), PtrNAC064 (XM_002319836; fgenesh1_pg.C_LG_XIII001004), and PtrNAC066 (XM_002329142; eugene3.01250035). The gene models for other poplar genes analyzed are as follows: gw1.I.9208.1 (PtrKNAT7), fgenesh1_pg.C_scaffold_124000096 (PtrNAC105), eugene3.00640212 (PtrNAC154), estExt_Genewise1_v1.C_LG_VII0653 (PtrNAC156), fgenesh1_pg.C_LG_IV000475 (PtrNAC157), eugene3.00110696 (PtrCesA8), estExt_Genewise1_v1.C_LG_XVI2679 (PtrGT43B), fgenesh1_pm.C_LG_IX000276 (PtrCCoAOMT1); those for PtrMYB genes were described by Wilkins et al. (2009). The Arabidopsis Genome Initiative locus identifiers for the Arabidopsis genes investigated in this study are as follows: MYB83 (At3g08500), MYB46 (At5g12870), SND1 (At1g32770), SND2 (At4g28500), SND3 (At1g28470), MYB20 (At1g66230), MYB42 (At4g12350), MYB43 (At5g16600), MYB52 (At1g17950), MYB54 (At1g73410), MYB58 (At1g16490), MYB63 (At1g79180), MYB69 (At4g33450), MYB85 (At4g22680), MYB103 (At1g63910), KNAT7 (At1g62990), NST1 (At2g46770), NST2 (At3g61910), VND6 (MAt5g62380), VND7 (At1g71930), CesA4 (At5g44030), CesA7 (At5g17420), CesA8 (At4g18780), FRA8 (At2g28110), IRX8 (At5g54690), IRX9 (At2g37090), 4CL1 (At1g51680), and CCoAOMT1 (At4g34050).

Acknowledgments

We thank Dr. G.A. Tuskan for sending us the stem cutting of poplar and Dr. Björn Sundberg for constructive comments and suggestions on the revision of the manuscript.

Notes

1This work was supported by the National Science Foundation (grant no. ISO–0744170) and the U.S. Department of Agriculture National Institute of Food and Agriculture AFRI Plant Biology program.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Zheng-Hua Ye (ude.agu.oibtnalp@eyhz).

[C]Some figures in this article are displayed in color online but in black and white in the print edition.

[OA]Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.148270

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