TY - JOUR
AU - Luo, Keming
AB - Abstract Wood is the most abundant biomass in perennial woody plants and is mainly made up of secondary cell wall. R2R3-MYB transcription factors are important regulators of secondary wall biosynthesis in plants. In this study, we describe the identification and characterization of a poplar MYB transcription factor PtoMYB92, a homolog of Arabidopsis MYB42 and MYB85, which is involved in the regulation of secondary cell wall biosynthesis. PtoMYB92 is specifically expressed in xylem tissue in poplar. Subcellular localization and transcriptional activation analysis suggest that PtoMYB92 is a nuclear-localized transcriptional activator. Overexpression of PtoMYB92 in poplar resulted in an increase in secondary cell wall thickness in stems and ectopic deposition of lignin in leaves. Quantitative real-time PCR showed that PtoMYB92 specifically activated the expression of lignin biosynthetic genes. Furthermore, transient expression assays using a β-glucuronidase (GUS) reporter gene revealed that PtoMYB92 is an activator in the lignin biosynthetic pathway during secondary cell wall formation. Taken together, our results suggest that PtoMYB92 is involved in the regulation of secondary cell wall formation in poplar by controlling the biosynthesis of monolignols. Introduction Lignin is most commonly derived from wood, and is an integral part of the secondary thickened plant cell walls. It is a complex polymer of aromatic alcohols known as monolignols, including coniferyl alcohol, sinapyl alcohol and coumaryl alcohol (Boerjan et al. 2003, Ralph et al. 2006, Vanholme et al. 2010). The monolignol biosynthetic pathway starts with the general phenylpropanoid pathway, resulting in the production of hydroxycinnamoyl-CoA esters, which are the common precursors of lignin and diverse groups of various phenolic compounds. To date, this biosynthetic pathway has been well established and a number of limiting enzymes have been identified (Baucher et al. 2003, Boerjan et al. 2003, Chen and Dixon 2007, Vanholme et al. 2008, Grabber et al. 2010, Novaes et al. 2010, Rahantamalala et al. 2010, Vanholme et al. 2010). Recent studies have revealed that expression of lignin biosynthetic genes is under the control of a common transcriptional network activating the entire secondary wall biosynthetic pathway (Zhong et al. 2011, Zhong and Ye 2014a). In the past decade, molecular and genetic studies have demonstrated that the co-ordinated activation of the secondary wall biosynthetic program is regulated by the secondary wall NAC (NAM, ATAF1/2 and CUC2)- and MYB-mediated transcriptional network (Zhong and Ye 2014). In the network, several closely related NAC domain transcription factors, including SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1), NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), NST2, VASCULARRELATED NAC DOMAIN6 (VDN6) and VND7, have been identified as master regulators that are able to activate the entire biosynthetic pathways of the secondary wall components, including cellulose, xylan and lignin (Mitsuda et al. 2007, Zhong and Ye 2007, Zhong et al. 2008). These NAC domain transcription factors can directly bind to the 19 bp SNBEs (secondary wall NAC-binding elements), which are present in the promoters of several wood-associated transcription factors, such as MYBs and BLH6, and a number of structural genes involved in secondary wall biosynthesis (Zhong and Ye 2010). In addition to NAC transcription factors, many R2R3-MYB transcription factors, which contain a highly conserved N-terminal DNA-binding domain and a highly variable C-terminal activation or repression domain, have also been identified as key regulators of secondary wall biosynthesis in plants (Rogers and Campbell 2004). In Arabidopsis thaliana, it has been well demonstrated that AtMYB46 and AtMYB83 as direct targets of secondary wall NACs, including SND1/NSTs/VNDs, function redundantly as master switches in the regulation of secondary wall biosynthesis (Zhong et al. 2007, Zhong and Ye 2012). AtMYB58, AtMYB63 and AtMYB85 are specific transcriptional activators of lignin biosynthesis in the SND1-mediated transcriptional network during secondary cell wall formation in Arabidopsis (McCarthy et al. 2009, Zhou et al. 2009). Other MYB transcription factors, such as AtMYB20, AtMYB52 and AtMYB54, play important roles in regulating secondary wall thickening (Zhong et al. 2008, McCarthy et al. 2009). In tree species, some wood-associated MYB transcription factors have also been well characterized so far. For example, PtMYB1, PtMYB4 and PtMYB8 from pine are involved in regulating secondary xylem formation (Patzlaff et al. 2003, Bomal et al. 2008). EgMYB2 from Eucalyptus, a homolog of AtMYB46 and AtMYB83, has been shown to alter phenylpropanoid metabolism, leading to ectopic deposition of lignin, when constitutively expressed in tobacco (Goicoechea et al. 2005). With the release of the Populus trichocarpa genome sequence (Tuskan et al. 2006), Populus has become a good model system for studying wood development, especially secondary cell wall formation. Compared with Arabidopsis, a similar but more complicated regulatory mechanism has been preliminarily identified in poplar (Zhong et al. 2011, Zhong and Ye 2012, Zhong and Ye 2014). There are at least 192 annotated genes encoding R2R3-MYB transcription factors in the Populus genome (Wilkins et al. 2009); however, only a few members of this family have been discovered and functionally characterized. For example, four MYB transcription factors, PtrMYB2, PtrMYB21, PtrMYB3 and PtrMYB20, have been demonstrated to be direct targets of wood-associated NAC domain transcription factors PtrWND2B and PtrWND6B (Zhong et al. 2011), and able to bind the secondary wall MYB-responsive elements (SMREs) in the promoters of their target genes to activate the biosynthetic pathways of cellulose, xylan and lignin (Zhong and Ye 2012), suggesting that they act as second-level master switches regulating secondary wall biosynthesis during wood formation in poplar. PtrMYB3 and PtrMYB20, which are functional orthologs of Arabidopsis MYB46 and MYB83, activated the expression of many secondary wall-associated transcription factors, including MYB42, MYB54, MYB58, MYB63 and MYB85 (McCarthy et al. 2010). Two other lignin-associated MYB transcription factors (PtoMYB216 and PtrMYB152) have also been isolated from poplar. Overexpression of PtoMYB216 specifically activated the expression of the upstream genes in the lignin biosynthetic pathway and resulted in ectopic deposition of lignin in transgenic poplar (Tian et al. 2013). PtrMYB152, a poplar homolog of Arabidopsis MYB43 and MYB20, has been shown to be a transcriptional activator and is involved in the regulation of secondary cell wall biosynthesis in poplar (Li et al. 2014, Wang et al. 2014). Six poplar MYB genes (MYB10/128, MYB90/167 and MYB92/125) were heterologously expressed in transgenic Arabidopsis and shown to play important roles in the regulation of secondary wall formation and control of flowering timing (Chai et al. 2014). More recently, ectopic expression of PdMYB221 from P. deltoides resulted in reduced cell wall thicknesses of fibers and vessels in Arabidopsis inflorescence stems, indicating that PdMYB221 is involved in the negative regulation of secondary wall formation through the direct and indirect suppression of the expression of genes involved in secondary wall biosynthesis (Tang et al. 2015). However, the exact functions of these MYB transcription factors in wood formation in poplar remain largely unknown. In this study, we report the functional characterization of the R2R3-MYB transcription factor PtoMYB92 which is isolated from Chinese white poplar (P. tomentosa Carr.). Phylogenetic analysis shows that PtoMYB92 is a poplar homolog of Arabidopsis MYB42 and MYB85. Overexpression of PtoMYB92 in transgenic poplar resulted in a dramatic increase in secondary cell wall thickness in fibers, accompanied by elevated expression of structural genes in the lignin biosynthetic pathway. Our findings indicate that PtoMYB92 is involved in the regulation of lignin biosynthesis during secondary cell wall formation in poplar. Results Isolation and characterization of PtoMYB92 from P. tomentosa To elucidate the functional role of MYB92 in secondary cell wall formation in poplar, a putative R2R3-MYB transcription factor gene, PtoMYB92, was isolated from P. tomentosa by reverse transcription–PCR (RT–PCR) according to the P. trichocarpa homolog. PtoMYB92 appeared to be a full-lengh cDNA of 813 bp encoding a protein of 270 amino acid residues, with a predicted molecular weight of about 30.6 kDa and a calculated isoelectric point (pI) of 5.09. Sequence analysis showed that PtoMYB92 contains two adjacent and highly conserved R2R3 repeats in the DNA-binding domain in the N-terminal region, while the C-terminal region shows limited homology to other known MYB proteins (Fig. 1A). Comparison of the R2R3 domain regions identified that the product of PtoMYB92 is closely related to other MYB proteins from other species and shares high homology with PbMYB (95.76%, similarity) in Pimpinella brachycarpa, AtMYB42 (94.92%, similarity), AtMYB85 (92.37%, similarity) and AtMYB43 (86.44%, similarity) in Arabidopsis, OsMYB14 (85.59%, similarity) in Oryza sativa and PtMYB1 (80.51%, similarity) in Pinus taeda L. (Fig. 1A). Fig. 1 View largeDownload slide Multiple sequence alignment and phylogenetic analysis of the amino acid sequences of PtoMYB92 with other R2R3-MYB transcription factors from various plant species. (A) Sequence similarity of PtoMYB92 including the conserved R2R3 DNA-binding domains with other MYB transcription factors. Amino acid sequences were aligned with DNAMAN software. Numbers on the left refer to amino acid sequence positions. The conserved R2R3-MYB domain is underlined. Identical and similar amino acid residues are shaded with black and gray, respectively. (B) The phylogenetic relationship of PtoMYB92 to other secondary wall-associated MYB transcription factors was constructed by the Neighbor–Joining method of MEGA5.1 software. The bootstrap values are shown as percentages at the nodes. Bar = 0.1 substitutions per site. The GenBank accession numbers of MYB transcription factors sequences are given in the Materials and Methods. Fig. 1 View largeDownload slide Multiple sequence alignment and phylogenetic analysis of the amino acid sequences of PtoMYB92 with other R2R3-MYB transcription factors from various plant species. (A) Sequence similarity of PtoMYB92 including the conserved R2R3 DNA-binding domains with other MYB transcription factors. Amino acid sequences were aligned with DNAMAN software. Numbers on the left refer to amino acid sequence positions. The conserved R2R3-MYB domain is underlined. Identical and similar amino acid residues are shaded with black and gray, respectively. (B) The phylogenetic relationship of PtoMYB92 to other secondary wall-associated MYB transcription factors was constructed by the Neighbor–Joining method of MEGA5.1 software. The bootstrap values are shown as percentages at the nodes. Bar = 0.1 substitutions per site. The GenBank accession numbers of MYB transcription factors sequences are given in the Materials and Methods. In previous studies, a number of R2R3-MYB transcriptions factors have been reported to participate in the regulation of secondary cell walls in plants (McCarthy et al. 2010, Zhao et al. 2013, Li et al. 2014). The phylogenetic relationship among conserved R2R3 repeat domains of secondary cell wall regulatory MYB transcription factors from poplar and other species was analyzed by Neighbor–Joining methods (Fig. 1B). Phylogenetic analysis revealed that PtoMYB92 and PtrMYB125 are grouped together with Arabidopsis MYB42 and MYB85, which have been demonstrated to participate in secondary cell wall formation (Zhong et al. 2008), as well as two other poplar MYB members (PtrMYB075 and PtrMYB199) (Fig. 1B). The results suggest that PtoMYB92 is a potential transcriptional regulator in secondary cell wall formation in poplar. Expression analysis of PtoMYB92 To determine the expression profiles of the PtoMYB92 gene, total RNA was extracted from roots, young leaves, old leaves, stems, xylem, phloem and petioles from 3-month-old poplar. Quantitative real-time PCR (qRT-PCR) analysis revealed that PtoMYB92 is expressed at a high level in xylem and stems, and at a moderate level in phloem, petioles, roots and old leaves, whereas the lowest level of expression is found in young leaves (Fig. 2). Fig. 2 View largeDownload slide Expression patterns of PtoMYB92 in poplar. Quantitative RT-PCR analysis of PtoMYB92 transcripts in different tissues of wild-type poplar. The poplar Actin gene was used an internal control. Error bars represent ± SD from three biological repeats. R, root; X, xylem; YL, young leaf; S, stem; Ph, phloem; OL, old leaf; Pe, petiole. Fig. 2 View largeDownload slide Expression patterns of PtoMYB92 in poplar. Quantitative RT-PCR analysis of PtoMYB92 transcripts in different tissues of wild-type poplar. The poplar Actin gene was used an internal control. Error bars represent ± SD from three biological repeats. R, root; X, xylem; YL, young leaf; S, stem; Ph, phloem; OL, old leaf; Pe, petiole. To investigate further the spatial and temporal expression pattern of PtoMYB92, a binary vector containing the GUS (β-glucuronidase) gene driven by the 1,638 bp promoter region of PtoMYB92 was transformed into Arabidopsis plants. Histochemical GUS staining analysis showed that GUS activity was detected in all tissues of 2-week-old transgenic plants, especially in stems (Supplementary Fig. S2A). In mature transgenic Arabidopsis plants, GUS staining was observed in flowers, stems and leaves. In the inflorescence, GUS activity was detected preferentially in vascular tissues of the stamens, anthers and petals (Supplementary Fig. S2B). Furthermore, GUS expression was observed in the vascular bundles and interfascicular fibers in the inflorescence stems (Supplementary Fig. S2C) and in the vascular vein of leaves (Supplementary Fig. S2D). These results indicated that PtoMYB92 was expressed preferentially in the tissues undergoing secondary cell wall thickening. PtoMYB92 acts as a transcriptional activator and is located in the nucleus In order to investigate the subcellular localization of PtoMYB92 in vivo, a construct containing the coding sequence of PtoMYB92 fused to the GFP (green fluorescent protein) reporter gene driven by the Cauliflower mosaic virus (CaMV) 35S promoter was transformed into tobacco epidermal cells via the particle bombardment method. As shown in Fig. 3A, tobacco cells expressing a control 35S-GFP gene showed a fluorescent signal in both the cytoplasm and nucleus, while the PtoMYB92:GFP fusion protein was only detected in the nucleus, consistent with functional localization of PtoMYB92 as a transcription factor. Fig. 3 View largeDownload slide Nuclear localization and transcriptional activity assay of PtoMYB92. (A) The PtoMYB92 protein was localized in the nucleus of tobacco leaf epidermal cells. GFP fluorescent images were examined with a confocal microscope at 18 h after bombardment. The position of the nucleus was confirmed by DAPI staining, and bright-field images were compared. (B) PtoMYB92 has trans-activation activity in yeast. The yeast carrying GAL4BD-PtoMYB92 can grow on medium without adenine, histidine and tryptophan (SD/–AHT) and induce the activity of X-β-gal, while the negative control cannot. Fig. 3 View largeDownload slide Nuclear localization and transcriptional activity assay of PtoMYB92. (A) The PtoMYB92 protein was localized in the nucleus of tobacco leaf epidermal cells. GFP fluorescent images were examined with a confocal microscope at 18 h after bombardment. The position of the nucleus was confirmed by DAPI staining, and bright-field images were compared. (B) PtoMYB92 has trans-activation activity in yeast. The yeast carrying GAL4BD-PtoMYB92 can grow on medium without adenine, histidine and tryptophan (SD/–AHT) and induce the activity of X-β-gal, while the negative control cannot. To determine whether PtoMYB92 is a transcriptional activator, the full-length sequence of PtoMYB92 was fused with the GAL4 DNA-binding domain coding sequence and transformed into the yeast strain Y2HGold. The yeast transformants harboring the GAL4:PtoMYB92 fusion gene grew well on the selection medium lacking leucine, histidine and adenine, and these cells turned blue when induced by the expression of the β-galactosidase reporter gene (Fig. 3B). These results indicate that PtoMYB92 is targeted to the nucleus and acts as a transcriptional activator. Overexpression of PtoMYB92 causes ectopic deposition of lignin in transgenic poplars To investigate its function in the regulation of secondary cell wall biosynthesis, we overexpressed PtoMYB92 under the control the CaMV 35S promoter in P. tomentosa Carr. More than 20 putative transgenic plants were generated, and the successful integration of the transgenes into the genome of transgenic plants was confirmed by PCR with gene-specific primers for the HPT (hygromycin phosphotransferase) gene (Supplementary Fig. S3). Three independently transformed lines with high expression of the PtoMYB92 gene were selected for in-depth characterization (Fig. 4A; Supplementary Fig. S4). Transgenic poplar lines overexpressing PtoMYB92 showed reduced plant height (Fig. 4B, C) and slightly curled leaves with smaller size (Fig. 4D) compared with the wild type. Similar phenotypes were also observed in Arabidopsis MYB46 overexpressors (Zhong et al. 2007). This finding suggested that overexpression of PtoMYB92 might induce ectopic deposition of lignin in transgenic plants. Fig. 4 View largeDownload slide Phenotypes of transgenic poplar plants overexpressing PtoMYB92. (A) Relative expression level of PtoMYB92 in three independent transgenic lines. The poplar Actin gene was used as an internal control. (B) Representative 8-month-old control and PtoMYB92 overexpression lines (L7, L16 and L23). (C) The heights of the control and PtoMYB92 overexpressors (L7, L16 and L23). (D) The leaf morphology of the control and PtoMYB92 overexpression plants. Measurements were made during 8 months of growth in the greenhouse. Data are means ± SD from at least three lines. Student’s t-test: *P < 0.05; **P < 0.01. Fig. 4 View largeDownload slide Phenotypes of transgenic poplar plants overexpressing PtoMYB92. (A) Relative expression level of PtoMYB92 in three independent transgenic lines. The poplar Actin gene was used as an internal control. (B) Representative 8-month-old control and PtoMYB92 overexpression lines (L7, L16 and L23). (C) The heights of the control and PtoMYB92 overexpressors (L7, L16 and L23). (D) The leaf morphology of the control and PtoMYB92 overexpression plants. Measurements were made during 8 months of growth in the greenhouse. Data are means ± SD from at least three lines. Student’s t-test: *P < 0.05; **P < 0.01. Anatomical cross-sections of the sixth internode of 8-month-old poplar plants were prepared to determine whether the stem growth resulted from xylem development in 35S-PtoMYB92 transgenic lines. Histochemical staining revealed that the stem cross-sections of transgenic 35S-PtoMYB92 lines (Fig. 5A, B) generated more secondary xylem than did the wild-type plants (Fig. 5D, E). An increased number of lignified fiber cell layers were observed in 35S-PtoMYB92 plants (13 layers, Fig. 5B), while only seven layers were observed in wild-type plants (Fig. 5E). Similar results were also obtained in other internodes of transgenic plants (Supplementary Fig. S5). Additionally, examination of leaves showed that PtoMYB92 overexpressors exhibited stronger lignin staining in veins (Fig. 5C) than wild-type leaves (Fig. 5F). Fig. 5 View largeDownload slide Effects of PtoMYB92 overexpression on lignin biosynthesis during secondary cell wall formation in poplar. Stems from the sixth internode of 8-month-old plants were stained with phloroglucinol-HCl. (A and B) Stem cross-sections from transgenic poplars. (C) Leaves from transgenic poplars. (D and E) Stem cross-sections from wild-type plants. (F) Leaves from the wild-type plants. Ph, phloem; Xy, xylem. Scale bars: (A, D) = 500 µm; (B, E) = 200 µm; (C, F) = 2 mm. Fig. 5 View largeDownload slide Effects of PtoMYB92 overexpression on lignin biosynthesis during secondary cell wall formation in poplar. Stems from the sixth internode of 8-month-old plants were stained with phloroglucinol-HCl. (A and B) Stem cross-sections from transgenic poplars. (C) Leaves from transgenic poplars. (D and E) Stem cross-sections from wild-type plants. (F) Leaves from the wild-type plants. Ph, phloem; Xy, xylem. Scale bars: (A, D) = 500 µm; (B, E) = 200 µm; (C, F) = 2 mm. Confocal microscopy of lignin autofluorensence showed more phloem fibers in stem cross-sections of transgenic 35S-PtoMYB92 plants compared with the wild type (Fig. 6A). Meanwhile, transgenic 35S-PtoMYB92 plants showed ectopic lignin autofluorescence in pith cells (Fig. 6A). Examination of leaves revealed that epidermal cells of 35S-PtoMYB92 overexpressors exhibited strong lignin autofluorescence signals, whereas wild-type epidermis showed weak signals (Supplementary Fig. S6). Furthermore, the secondary cell wall thickness of vessels and xylary fibers of 35S-PtoMYB92 plants was dramatically increased as compared with the control lines (Fig. 6B). The vessel cell walls of PtoMYB92 overexpression plants were 30.6–43.8% thicker than those of the wild-type control (Supplementary Table S2). These results indicated that overexpression of PtoMYB92 induced secondary wall thickening, primarily the ectopic deposition of lignin. Fig. 6 View largeDownload slide Ectopic deposition of lignin in PtoMYB92-overexpressing transgenic poplar. (A) Laser scanning confocal microscopy of stem sections at the eighth internode in PtoMYB92-overexpressing transgenic poplar. (B) Scanning electron micrographs of stem cross-sections at the eighth internode in PtoMYB92-overexpressing transgenic poplar. Ph, phloem; Xy, xylem; Ve, vessel; Xf, xylary fiber. Scale bars: A = 200 µm, B = 10 µm. Fig. 6 View largeDownload slide Ectopic deposition of lignin in PtoMYB92-overexpressing transgenic poplar. (A) Laser scanning confocal microscopy of stem sections at the eighth internode in PtoMYB92-overexpressing transgenic poplar. (B) Scanning electron micrographs of stem cross-sections at the eighth internode in PtoMYB92-overexpressing transgenic poplar. Ph, phloem; Xy, xylem; Ve, vessel; Xf, xylary fiber. Scale bars: A = 200 µm, B = 10 µm. To examine the changes in the composition of the secondary cell wall, we measured the chemical components in PtoMYB92 overexpression lines and wild-type plants by both the acetyl bromide (AcBr) and Klason methods. The result showed that the Klason lignin contents were significantly increased by 14.04–28.1% in stems of the tested transgenic 35S-PtoMYB92 lines compared with the wild type. Similar results were also obtained when using the AcBr method (Supplementary Table S3). In addition, overexpression of PtoMYB92 appeared to have no remarkable effect on glucan contents, whereas lower xylan levels (9.64–17.1%) were detected in these transgenic lines compared with the wild-type plants (Supplementary Table S3). Overexpression of PtoMYB92 induces the expression of secondary cell wall biosynthetic genes in poplar Given that PtoMYB92 positively regulates secondary cell wall biosynthesis in poplar, we used qRT-PCR to determine the expression levels of the genes associated with secondary cell wall biosynthesis in transgenic plants. Of these, monolignol biosynthetic genes, including CCOAOMT1, CCR2, COMT2, C3H, PAL4, 4CL5, HCT1, C4H2 and CAD1, were significantly up-regulated in all transgenic plants overexpressing PtoMYB92 as compared with wild-type plants (Fig. 7A), indicating that PtoMYB92 positively regulated lignin biosynthesis in poplar. GT8D, GT43B and GT43D were down-regulated in transgenic plants (Fig. 7B), suggesting that PtoMYB92 might negatively affect xylan biosynthesis. Interestingly, the expression levels of CesA2B and CesA3A (Fig. 7B) showed no significant difference between 35S-PtoMYB92 plants and the wild-type control. These results were consistent with the quantification of secondary cell wall compositions of 35S-PtoMYB92 plants. Fig. 7 View largeDownload slide Gene expression analysis of secondary wall biosynthetic genes in PtoMYB92-overexpressing poplar plants. (A) Relative expression levels of genes involved in lignin biosynthesis (CCOAOMT1, CCR2, COMT2, C3H3, PAL4, HCT1, C4H2 and CAD1). (B) Relative expression levels of genes involved in the biosynthesis of lignin (4CL5), cellulose (CesA2B and CesA3A) and xylan (GT8D, GT43B and GT43D). The poplar Actin gene was used as an internal control. Error bars represent ± SD from three biological repeats. Student’s t-test: *P < 0.05; **P < 0.01. Fig. 7 View largeDownload slide Gene expression analysis of secondary wall biosynthetic genes in PtoMYB92-overexpressing poplar plants. (A) Relative expression levels of genes involved in lignin biosynthesis (CCOAOMT1, CCR2, COMT2, C3H3, PAL4, HCT1, C4H2 and CAD1). (B) Relative expression levels of genes involved in the biosynthesis of lignin (4CL5), cellulose (CesA2B and CesA3A) and xylan (GT8D, GT43B and GT43D). The poplar Actin gene was used as an internal control. Error bars represent ± SD from three biological repeats. Student’s t-test: *P < 0.05; **P < 0.01. To further identify the role of PtoMYB92 in the regulation of secondary wall biosynthesis, the promoters of six secondary wall biosynthetic genes, i.e. CCOAOMT1, CCR2 and C3H3 for lignin, GT8D and GT43B for xylan, and CesA2B for cellulose, were used to control GUS reporter gene expression (Fig. 8A). These reporter constructs were co-transfected with the transcription factor overexpression construct into Arabidopsis leaves for transactivation analysis. We found that PtoMYB92 induced the GUS reporter gene expression driven by the CCOAOMT1, CCR2 and C3H3 promoters by approximately 12.3-, 7.1- and 3.3-fold, and reduced the expression of GT8D and GT43B promoters by 0.29- and 0.41-fold compared with the corresponding controls (Fig. 8B), respectively. In addition, the expression of the CesA2B promoter was not activated by PtoMYB92. These results indicated that PtoMYB92 is a transcription activator of the lignin biosynthetic pathway during secondary cell wall formation, more specifically regulating lignin biosynthesis, while negatively affecting xylan biosynthesis in poplar. Fig. 8 View largeDownload slide Activation of the secondary cell wall biosynthetic gene promoters by PtoMYB92. (A) Diagrams of the effector and reporter constructs used for transactivation analysis. The effector construct is the PtoMYB92 cDNA driven by the 35S promoter. The reporter construct contains the GUS reporter gene driven by the promoters of poplar CCOAOMT1, CCR2, C3H3, GT8D, GT43B and CesA2B genes. (B) Transactivation analysis showing the PtoMYB92-activated expression of the GUS reporter gene driven by the promoter of CCOAOMT1, CCR2 and C3H3, and the PtoMYB92-repressed expression of the GUS reporter gene driven by the promoter of GT8D and GT43B. GUS activity in leaves transfected with the reporter construct pCXGUS-P vector alone was used as a control. Error bars represent ± SD of three biological replicates. Student’s t-test: *P < 0.05; **P < 0.01. Fig. 8 View largeDownload slide Activation of the secondary cell wall biosynthetic gene promoters by PtoMYB92. (A) Diagrams of the effector and reporter constructs used for transactivation analysis. The effector construct is the PtoMYB92 cDNA driven by the 35S promoter. The reporter construct contains the GUS reporter gene driven by the promoters of poplar CCOAOMT1, CCR2, C3H3, GT8D, GT43B and CesA2B genes. (B) Transactivation analysis showing the PtoMYB92-activated expression of the GUS reporter gene driven by the promoter of CCOAOMT1, CCR2 and C3H3, and the PtoMYB92-repressed expression of the GUS reporter gene driven by the promoter of GT8D and GT43B. GUS activity in leaves transfected with the reporter construct pCXGUS-P vector alone was used as a control. Error bars represent ± SD of three biological replicates. Student’s t-test: *P < 0.05; **P < 0.01. Discussion Wood is the most abundant biomass on earth and is mainly composed of secondary cell wall. Therefore, it is vital to understand the molecular regulation mechanisms of secondary cell wall biosynthesis. In the past decade, numerous studies have demonstrated that secondary cell wall biosynthesis is controlled by a transcription factor network (Zhong et al. 2008, Zhong and Ye 2014). In this network, a number of R2R3-MYB transcription factors from Arabidopsis, Populus, Eucalyptus and pine have been identified as regulating secondary wall biosynthesis during wood formation (Patzlaff et al. 2003, Goicoechea et al. 2005, McCarthy et al. 2010, Tian et al. 2013, Wang et al. 2014, Zhong and Ye 2014). In poplar, there are at least 192 annotated R2R3-MYB transcription factors (Wilkins et al. 2009); however, only a few members involved in the regulation of secondary cell wall biosynthesis have been functionally characterized so far (McCarthy et al. 2010, Tian et al. 2013, Wang et al. 2014, Tang et al. 2015). In this study, we reported the characterization of PtoMYB92, a homolog of Arabidopsis MYB42 and MYB85 (Fig. 1B), in the regulation of secondary wall biosynthesis. The N-terminus of PtoMYB92 contained a conserved R2R3 repeat domain shared by AtMYB42 (Zhao and Dixon 2011), AtMYB43 (Ehlting et al. 2005), AtMYB85 (Nakano et al. 2010), OsMYB14 (Patzlaff et al. 2003), PbMYB (Ban et al. 2007) and PtMYB1 (Patzlaff et al. 2003) with high similarity (Fig. 1A). The PtoMYB92 gene is preferentially expressed in secondary xylem in poplar (Fig. 2). Overexpression of PtoMYB92 in transgenic plants resulted in ectopic deposition of lignin in the secondary cell wall (Figs. 5, 6) and the up-regulated expression of secondary wall biosynthetic genes (Fig. 7), suggesting its involvement in the regulation of wood formation in poplar. Previous studies have found that the Populus genome had undergone at least three rounds of genome-wide duplication followed by multiple segmental and tandem duplications (Tuskan et al. 2006, Chai et al. 2014). More than 80 pairs of paralogous R2R3-MYB genes in Populus were identified based on their chromosome placement and motif structures (Chai et al. 2014). In Arabidopsis, MYB46/83 act as master switches to regulate secondary wall biosynthesis genes for cellulose, xylan and lignin (Zhong et al. 2007) and concomitantly induce ectopic deposition of secondary wall components and reduce plant growth (Ko et al. 2009). Complementation and overexpression studies have demonstrated that four poplar MYB transcription factors (PtrMYB2/3/20/21) are orthologs of Arabidopsis MYB46/MYB83, and they are functionally conserved (Zhong et al. 2013). MYB46/83 also bind to the SMRE sites and activate expression of downstream transcription factors such as MYB42 and MYB85 (Zhong et al. 2008). By searching for poplar homologs of Arabidopsis MYB42 and MYB85 genes which are known to be associated with the regulation of secondary cell wall biosynthesis (Nakano et al. 2010, Zhao and Dixon 2011), four poplar R2R3-MYB transcription factors, i.e. PtrMYB75, PtrMYB92, PtrMYB125 and PtrMYB199, were identified (Fig. 1B). Consistent with our results as shown in Fig. 2, PdMYB92/125 (Arabidopsis MYB85 orthologs) were specifically expressed in xylem (Chai et al. 2014), indicating that their functions are involved in secondary wall formation. Ectopic overexpression of poplar PdMYB92/125 in Arabidopsis decreased stem fiber and vessel cell wall thickness (Chai et al. 2014). In our experiment, however, constitutive expression of PtoMYB92 in poplar activates the expression of lignin biosynthetic genes (Fig. 7) and induces ectopic deposition of lignin (Fig. 6; Supplementary Table S2). Our findings are consistent with a previous report in Arabidopsis showing that MYB85 specifically regulates the lignin biosynthetic pathway (Zhong et al. 2008). These results indicate that although the functions of some orthologous R2R3-MYB transcription factors from poplar and Arabidopsis appear to be conserved in regulating secondary wall biosynthesis, the transcriptional regulation network of secondary wall biosynthesis may be different in herbaceous and woody plants. Arabidopsis has been proved to be an excellent model for investigation of secondary cell wall formation. To date, an Arabidopsis gene regulatory network for secondary cell wall synthesis has been well established (Zhong et al. 2011, Zhong et al. 2014b). Although poplar has a more complex and multilevel transcriptional regulatory network during secondary cell wall formation (Andersson-Gunnerås et al. 2006), there exists a conserved transcription factor network that regulates secondary cell wall biosynthesis in poplar and Arabidopsis (Ohtani et al. 2011, Zhong et al. 2011, Zhong et al. 2013, Wang et al. 2014). Combining the experimental results in this study and previous works in poplar (Zhong et al. 2011, Zhong et al. 2013, Zhong et al. 2014), we speculate that PtoMYB92 may be directly and indirectly regulated by NAC master switches (PtrWNDs and PtrVNSs) and, in turn, PtoMYB92 specifically up-regulates the expression of the lignin biosynthetic genes. In summary, our study provides molecular evidence suggesting that PtoMYB92 acts as a positive regulator of secondary cell wall formation in vascular tissue, specifically activating lignin biosynthesis in poplar. The information in this study will be useful to design strategies for genetic improvement of wood biomass. Materials and Methods Plant materials Arabidopsis thaliana (ecotype Columbia; Col-0) plants were grown in an illuminated incubator at 22–23°C under a 16/8 h light/dark cycle with 10,000 lux of supplementary light, and the humidity of the illuminated incubator was approximately 80%. Populus tomentosa Carr. (clone 741) was grown in the greenhouse at 25°C under a 16/8 h light/dark cycle with 5,000 lux of supplementary light, and relative humidity was around 60% for optimum growth (Li et al. 2014). Cloning of full-length PtoMYB92 cDNA Total RNA was extracted from the leaf samples using plant Trizol Reagent (Tiangen) as described in the manufacturer’s instructions. The full-length cDNA fragment encoding PtoMYB92 was amplified with gene-specific primers (Supplementary Table S1) based on PtrMYB92 (XP_006368692) from P. trichocarpa by PCR. The PCR was carried out with Pfu DNA polymerase (TAKARA) in a total volume of 100 µl at 94°C for 4 min, 36 cycles of 94°C for 45 s, 58°C for 60 s and 72°C for 90 s, followed by a final extension of 72°C for 10 min. The total volume of the reagent was 100 µl, which contained 55 µl of GoTaqR Green Master Mix (Promega), 2.5 µl of each primer, 5 µl of cDNA as DNA template and 37.5 µl of nuclease-free water. PCR products were subcloned into the pMD19-T plasmid (TAKARA). After sequencing (BGI, Shenzhen, China), the PCR products were cloned into the plant binary vector pCXSN (Chen et al. 2009) between the CaMV 35S promoter and the nopaline synthase terminator (Tnos). The resulting vector p35S-PtoMYB92 with the HPT gene (Supplementary Fig. S1A), which confers resistance to hygromycin, was introduced into Agrobacterium tumefaciens strain EHA105 by the freeze–thaw method. Sequence alignment and phylogenetic analysis Database searches of the nucleotide and deduced amino acid sequences were performed through the NCBI/GenBank/Blast Sequence alignment (Altschul et al. 1990). Multiple sequence alignments were performed with DNAMAN. Phylogenic analyses were performed using MEGA version 5.1 (Lynnon Biosofta). qRT-PCR analysis Total RNA was extracted from roots, stems, young leaves, old leaves, phloem, xylem and petioles of poplar plants using the Trizol Reagent as described above. Then genomic DNA was removed from total RNA, and cDNA was synthesized using a PrimeScript™ RT reagent Kit with genome DNA Eraser (TAKARA). qRT-PCR was performed with a TP700 Real-Time PCR machine (TAKARA) using the SYBR Green PCR master mix kit (TAKARA) following the manual’s recommendations. The relative amount of gene expression was calculated using the expression of the poplar Actin gene as internal standard. Three biological replicates were performed for all of the samples and three technical replicates were carried out for each reaction. Gene-specific primers used in qRT-PCR assays are listed in Supplementary Table S1. Subcellular localization of PtoMYB92 The open reading frame (ORF) of PtoMYB92 was amplified with gene-specific primers (Supplementary Table S1) and cloned into the pCX-DG vector (Chen et al. 2009) to generate a 35S-PtoMYB92:GFP fusion vector (Supplementary Fig. 1B). Then the recombinant plasmid 35S-PtoMYB92:GFP and the control plasmid 35S-GFP were bombarded into tobacco (Nicotiana benthamiana) epidermal cells using Gene Gun GJ-1000 (SCIENTZ), respectively. The tobacco epidermis was stained with 4′,6-diamidino-2-phenylindole (DAPI), and observed with a confocal laser microscope (Leica TCS SP5). Yeast one-hybrid assay The full-length ORF of PtoMYB92 was amplified with specific primers (Supplementary Table S1), the amplification product was inserted into pGBKT7 (Clontech) and the recombinant plasmid was introduced into the yeast strain Saccharomyces cerevisiae Gold2 by the method described previously (Zaragoza et al. 2004). Transformants were grown on SD medium lacking Trp (tryptophan) for selection of positive clones and then on SD medium lacking Trp, His (histidine) and Ade (adenine) for the transactivation assay. In addition, X-α-gal was used to identify the transcription activation activity of PtoMYB92. Transformation of P. tomentosa Carr. plants Populus tomentosa Carr. plants were transformed by the Agrobacterium-mediated leaf disc method as described previously (Jia et al. 2010). In brief, leaf discs were infected with Agrobacterium cultures containing the p35S-PtoMYB92 construct and then were co-cultured with agrobacteria in Woody Plant Medium (WPM) in the dark for 3 d. The infected leaf discs were transferred to callus-inducing medium containing 2.0 mg l−1 zeatin, 1.0 mg l−1 1-naphthalene acetic acid (NAA), 400 mg l−1 cefotaxime, 9 mg l−1 hygromycin and 0.8% (w/v) agar. After about 3 weeks of culture in the dark, some calli were generated from the infected leaf discs of poplar. To induce adventitious buds, these leaf discs were transferred to selective medium, which contains 2.0 mg l–1 zeatin, 0.1 mg l–1 NAA, 400 mg l–1 cefotaxime, 9 mg l–1 hygromycin and 0.8% (w/v) agar. Regenerated shoots were transferred to rooting medium containing 0.1 mg l–1 NAA, 400 mg l–1 cefotaxime and 9 mg l–1 hygromycin. Rooted plantlets were acclimatized in pots and then grown in the greenhouse. The presence of the transgenes in transformed poplar plants was detected by PCR with gene-specific primers for the HPT gene (Supplementary Table S1). Analysis of the PtoMYB92 promoter The promoter fragment of PtoMYB92 was amplified from the genomic DNA of P. tomentosa Carr. by the gene-specific primers which are listed in Supplementary Table S1. The amplified promoter fragment (1,638 bp) was cloned into the pCXGUS-P vector (Chen et al. 2009) to produce the proPtoMYB92-GUS construct (Supplementary Fig. 1C). The construct was then transferred into A. tumefaciens strain EHA105 for transformation of Arabidopsis. The floral dip method was used to transform it into wild-type Arabidopsis (Clough and Bent 1998). Transformants were selected on Murashige and Skoog (MS) medium with 50 mg l–1 hygromycin. Seedlings which survived were transferred into the soil and grown in an illuminated incubator. For histochemical GUS staining (Jefferson 1987), transgenic seedlings were fixed in acetone for 10 min at 4°C, washed three times with 100 mm NaPO4 buffer (pH 7.0) and incubated with GUS staining solution [100 mm NaPO4 (pH 7.0), 10 mm EDTA, 2 mm 5-bromo-4-chloro-3-indolyl-β-GlcA, 5 mm K4Fe(CN)6, 5 mm K3Fe(CN)6 and 0.2% Triton X-100] for 3 h at 37°C. The reaction was stopped, and Chl was extracted using 75% ethanol. More than five transgenic seedlings were used for GUS staining. All experiments were repeated at least three times. Transient expression and GUS activity assay The promoter fragments of secondary cell wall biosynthetic genes were amplified by PCR with the gene-specific primers described in Supplementary Table S1. These fragments were individually fused to the GUS reporter gene in the pCXGUS-P vector (Chen et al. 2009) to generate reporter constructs (Supplementary Fig. 1D–J). Agrobacterium cells carrying the 35S-PtoMYB92 construct were used as an effector. The leaves of transgenic Arabidopsis transformants containing 35S-PtoMYB92 were infiltrated by the reporters with the agroinfiltration method (Kim et al. 2009). After 3 d of infiltration, GUS activity was quantitatively measured by spectrophotometry (Jefferson et al. 1987). Scanning electron microscopy analysis For scanning electron microscopy (SEM), transverse sections were hand-cut on stems (the eighth internode) of 8-month-old poplar plants. Stem cross-sections were dissected transversely with razor blades. Wood samples were attached to the scanning electron microscope using double-sided sticky tape. Samples were directly viewed under the microscope (Phenom™ Pure FEI) following the manual’s recommendations, and images were captured digitally. For each line, six areas were randomly selected and images were taken to measure the vessel cell wall thickness (µm) using the software Revolution 1.6.1. Histochemical staining of lignin For histochemical staining, stem (from the fourth to sixth internode) sections from 8-month-old plants grown in the greenhouse were hand-cut with a razor blade and an Ultra-Thin Semiautomatic Microtome (FINESSE 325, Thermo). The microsections and leaves were stained for 15 s with 1.0% (w/v) phloroglucinol after dissociation for 60 s by 40% (v/v) HCl (hydrochloric acid), and then observed under an Olympus BX53 microscope (Qin et al. 2007). Digital images were captured with a diagnostic instrument and processed using the software Adobe Photoshop CC. Determination of lignin autofluorescence Transverse sections were hand-cut on poplar stems (the eighth internode) of 8-month-old plants. Stem cross-sections was dissected transversely with an Ultra-Thin Semiautomatic Microtome (FINESSE 325, Thermo). Wood samples were attached using double-sided sticky tape and directly viewed under a confocal laser microscope (Leica TCS SP5) following the manual’s recommendations, and images were captured digitally. Chemical analysis of secondary cell wall components Wood particles (40 mesh) were screened using a mill and then extracted with benzene/ethanol (2 : 1, v/v) for 8 h. The resulting wood meals were used for lignin content determination to estimate both Klason lignin and acid-soluble lignin for total lignin content (Dence 1992). Briefly, 0.2 g of samples was hydrolyzed with 3 ml of 72% (w/w) H2SO4 at 20°C for 2 h. The mixture was diluted with 100 ml of water and autoclaved at 121°C for 1 h, and then filtered through a fine coarseness crucible and dried overnight at 105°C. Acid-insoluble lignin was determined gravimetrically. The filtrates were diluted with water to 250 ml to determine the amount of acid-soluble lignin at 205 nm (Shimadzu UV-2401 PC UV-VIS). The AcBr-soluble lignin method was employed as another way to estimate the total lignin content, as described by Iiyama and Wallis (1988). The cellulose and xylan contents were estimated by the Van Soest method (Van Soest and Wine 1967). The extracted sample (0.2 g) was digested with 20 ml of acid detergent solution (0.5 M H2SO4, 20 g l–1 CTAB) for 1 h at 100°C, filtrated with a vacuum, and then washed three times with hot distilled water. After being washed twice with acetone, the crucibles were dried overnight at 100°C and weighed. The filtrates were diluted with water to 250 ml to determine the amount of acid-soluble lignin at 530 nm (Shimadzu UV-2401 PC UV-VIS). Statistical analysis The Student’s t-test program (http://www.graphpad.com/quickcalcs/ttest1.cfm) was used for statistical analysis of all measurement data. The quantitative differences between two groups of data for comparison in all these experiments were shown to be statistically significant (P < 0.05) GenBank accession numbers for genes used in this study The accession number of PtoMYB92 in the GenBank database is KP710214. Other GenBank accession numbers for genes used in this study are as follows: PtrCCOAOMT1 (EU603307.1), PtrCCR2 (EU603310.1), PtrCOMT2 (EU603317.1), PtrC3H3 (EU603301.1), PtrPAL4 (EU603322.1), PtrHCT1 (EU603313.1), PtrC4H2 (EU603302.1), PtrCAD1 (EU603306.1), Ptr4CL5 (EU603299.1), PtrF5H2 (EU603311.1), PtrCesA2B (JX552008.1), PtrCesA3A (JX552264.1), PtrGT8D (EF501824.1), PtrGT43B (JF518935.1), PtrGT43D (JF518937.1), AtMYB4 (AEE86955.1), AtMYB42 (AF175999.1), AtMYB43 (AF175990.1), AtMYB58 (AF062893.1), AtMYB61 (AF062896.1), AtMYB63 (AF062898.1), AtMYB85 (AF175993.2), PbMYB (AF161711.1), OsMYB14 (Y11351.1), PtMYB1 (AY356372.1), GhMYB1 (L04497.1), GhMYB6 (AF034134.1), HvMYB3 (X70881.1), EgMYB1 (AJ576024.1), EgMYB2 (AJ576023.1), ZmMYB31 (AM156906.1), and ZmMYB42 (AM156908.1), PtrMYB75 (XP_002321838.2), PtrMYB125 (XP_002303526.2), PtrMYB199 (XP_002318878.1) and PtrMYB216 (XP_002319449.1). Funding This work was supported by the National Natural Science Foundation of China [31370672 and 31171620 to K. L., 31300990 to D. F. ]; The Chinese Academy of Sciences [One hundred Talents Program to K. L.]; the Natural Science Foundation Project of CQ [CSTC2013JJB8007 to K. L.]; the Fundamental Research Funds for the Central Universities [XDJK2014a005 to K. L., XDJK2014C062 to D. F.]. Disclosures The authors have no conflicts of interest to declare. Abbreviations Abbreviations AcBr acetyl bromide CaMV Cauliflower mosaic virus GFP green fluoresent protein GUS β-glucuronidase HPT hygromycin phosphotransferase ORF open reading frame qRT-PCR quantitative real-time PCR WT wild type References Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. ( 1990) Basic local alignment search tool. J. Mol. Biol.  215: 403– 410. Google Scholar CrossRef Search ADS PubMed  Andersson-Gunnerås S. Mellerowicz E.J. Love J. Segerman B. Coutinho P.M. Nisson P. et al.   . 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TI - PtoMYB92 is a Transcriptional Activator of the Lignin Biosynthetic Pathway During Secondary Cell Wall Formation in Populus tomentosa
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcv157
DA - 2015-10-27
UR - https://www.deepdyve.com/lp/oxford-university-press/ptomyb92-is-a-transcriptional-activator-of-the-lignin-biosynthetic-zmxKMkGDZ9
SP - 2436
EP - 2446
VL - 56
IS - 12
DP - DeepDyve
ER -