PtrWOX13A Promotes Wood Formation and Bioactive Gibberellins Biosynthesis in Populus trichocarpa

Abstract

WUSCHEL-related homeobox (WOX) genes are plant-specific transcription factors (TFs) involved in multiple processes of plant development. However, there have hitherto no studies on the WOX TFs involved in secondary cell wall (SCW) formation been reported. In this study, we identified a Populus trichocarpa WOX gene, PtrWOX13A, which was predominantly expressed in SCW, and then characterized its functions through generating PtrWOX13A overexpression poplar transgenic lines; these lines exhibited not only significantly enhanced growth potential, but also remarkably increased SCW thicknesses, fiber lengths, and lignin and hemicellulose contents. However, no obvious change in cellulose content was observed. We revealed that PtrWOX13A directly activated its target genes through binding to two cis-elements, ATTGATTG and TTAATSS, in their promoter regions. The fact that PtrWOX13A responded to the exogenous GAs implies that it is responsive to GA homeostasis caused by GA inactivation and activation genes (e.g., PtrGA20ox4, PtrGA2ox1, and PtrGA3ox1), which were regulated by PtrWOX13A directly or indirectly. Since the master switch gene of SCW formation, PtrWND6A, and lignin biosynthesis regulator, MYB28, significantly increased in PtrWOX13A transgenic lines, we proposed that PtrWOX13A, as a higher hierarchy TF, participated in SCW formation through controlling the genes that are components of the known hierarchical transcription regulation network of poplar SCW formation, and simultaneously triggering a gibberellin-mediated signaling cascade. The discovery of PtrWOX13A predominantly expressed in SCW and its regulatory functions in the poplar wood formation has important implications for improving the wood quality of trees via genetic engineering.

Keywords: Populus trichocarpa; PtrWOX13A; bioactive gibberellins biosynthesis; coordinated regulation; wood formation.

FIGURE 1

Tissue-specific expression patterns, subcellular localization, and transcriptional activity of PtrWOX13A. (A) The tissue-specific expression patterns of three PtrWOX13 genes as determined by qRT-PCR analysis. PtrActin was used as a reference gene. The expression of PtrWOX13A in root was set to 1 and expression in all other tissues is relative to roots. (B) The temporal expression patterns of three PtrWOX13 genes after GA treatments as determined by qRT-PCR analysis. PtrActin was used as a reference gene. The expression of PtrWOX13A at 0 h was set to 1, all other time points were relative to 0 h. (C) Subcellular localization of PtrWOX13A. Confocal images manifested the localization of PtrWOX13A: DAPI, a nuclear staining dye; GFP proteins in the nuclei of onion epidermal cells. Merge: The merged images of White light, DAPI, and GFP staining. Arrows indicate cells located in the epidermis of onions. (D) Transcriptional activation analysis of PtrWOX13A fused with the GAL4 DNA binding domain (GAL4DB) in yeast showed its potential to activate the expression of the His-3 and X-α-Gal reporter genes. Arrows indicate cells located in the epidermis of onions.

FIGURE 2

Effect of PtrWOX13A overexpression on growth-related traits in Populus trichocarpa transgenic lines. (A) Average leaf length and leaf width; (B) Average height and number of stem nodes and standard error of each transgenic line; (C) Average fresh weight and dry weight; (D) Average base diameters and breaking forces. All the data were measured on the 90-day-old wild type (WT) and PtrWOX13A transgenic lines (OE-2, OE-4, and OE-7). Each error bar represents the standard deviation of three biological replicates. Asterisks denote levels of significance (Dunnett’s test; *p < 0.05 significant, and **p < 0.01, highly significant).

FIGURE 3

Effect of PtrWOX13A overexpression on the secondary wall thickness of stems in Populus trichocarpa. (A–F) The scanning electron microscope of cross stem sections of wild type (A–C) and PtrWOX13A overexpression transgenic lines (D–F). (A,D) represent 34×, (B,E) represent 1,500×, and (C,F) represent 5,000× scanning electron microscope of cross stem sections, respectively. V and F in (B,C,E,F) represent vessel cell and fiber cell, respectively. (G) The fiber wall thicknesses of cross stem sections in wild type (WT) and PtrWOX13A overexpression transgenic lines (OE-2, OE-4, and OE-7). (H) The vessel wall thicknesses of cross stem sections in WT and PtrWOX13A overexpression transgenic lines. Error bars represent the standard deviation of three biological replicates. Asterisks indicate levels of significance (Dunnett’s test; **p < 0.01). All data were measured on the 8th stem of 90-day-old poplars.

FIGURE 4

Impact of PtrWOX13A overexpression on components of the secondary cell wall of stems in Populus trichocarpa. (A) Stem section of wild type stained with Phloroglucinol-HCl (red color). (B) Stem section of wild type stained with Calcofluor white staining (blue color). (C) Stem section of wild type stained with Monoclonal Antibody LM10 (green color). (D) Stem section of PtrWOX13A overexpression transgenic lines stained with Phloroglucinol-HCl (red color). (E) Stem section of PtrWOX13A overexpression transgenic lines stained with Calcofluor white (blue color). (F) Stem section of PtrWOX13A overexpression transgenic lines stained with Monoclonal Antibody LM10 (green color). Scale bars = 100 μm. All data were measured on the 8th stem of 90-day-old poplars.

FIGURE 5

Effect of PtrWOX13A overexpression on secondary cell wall of stems in Populus trichocarpa. (A–E) Represent lignin content, cellulose content, hemicellulose content, fiber length, fiber diameter, GA₁ content, and GA₄ content, respectively. Error bars represent the standard deviation of three biological replicates. Asterisks indicate levels of significance (Dunnett’s test; *p < 0.05 significant, and **p < 0.01, highly significant).

FIGURE 6

Expression analysis of wood formation pathway genes and their regulatory genes in 90 days old wild-type and PtrWOX13A transgenic lines. (A) Represents expression analysis of lignin biosynthesis genes, cellulose biosynthesis genes, and hemicellulose biosynthesis genes. (B) Represents expression of fiber cell elongation genes, GA biosynthesis genes, and TFs. PtrActin was used as a reference gene. Control represents the normalized expression level (namely 0 in this case) of each gene examined in wild type plants. Each error bar represents the standard deviation of three biological replicates. Asterisks indicate levels of significance (Dunnett’s test; **p < 0.01, highly significant). Those genes include lignin biosynthesis genes (PtrPAL4, PtrC4H1, PtrC3H3, Ptr4CL5, PtrCCoAOMT3, PtrCOMT2, PtrCCR2, PtrCAld5H2, and PtrCAD1), lignin polymerization genes (PtrLAC19, PtrLAC21, PtrLAC26, and PtrLAC41), cellulose biosynthesis genes (PtrCESA4, PtrCESA7, and PtrCESA8), hemicellulose biosynthesis genes (PtrGT43A, PtrGT47C, and PtrGT8F), fiber cell elongation genes (PtrCSLD2, PtrXTH5, PtrEXPA8, and PtrFRA2), GA biosynthesis genes (PtrGA20ox4, PtrGA3ox1, and PtrGA2ox1), and TFs (PtrWND6A, PtrWND6B, PtrMYB20, PtrMYB21, PtrMYB28, PtrMYB152, PtrMYB157, PtrMYB52, and PtrMYB54).

FIGURE 7

Activation of the promoters of poplar transcription factors and secondary cell wall biosynthesis pathway genes by PtrWOX13A. (A,B) Diagrams of the effector and the reporter constructs. (C) The expression of the GUS under the control of those promoters that were activated by PtrWOX13A. GUS activity in tobacco leaves transfected with pROKII empty vector, reporter vector, and 35S-LUC-pGreenII 8000 vector was used as a control and was set to 1. GUS activity for each promoter tested is expressed as the ratio of GUS/LUC obtained with the effector pROKII-PtrWOX13A to GUS/LUC obtained with the control effector pROKII empty vector. Error bars represent the standard deviation of three biological replicates. Asterisks indicate levels of significance of differential expression (Dunnett’s test; **p < 0.01, highly significant). Those genes include lignin biosynthesis genes (PtrC4H1, PtrC3H3, Ptr4CL5, PtrCCoAOMT3, and PtrCCR2), hemicellulose biosynthesis genes (PtrGT8F), fiber cell elongation genes (PtrXTH5 and PtrEXPA8), GA biosynthesis genes (PtrGA20ox4, PtrGA3ox1, and PtrGA2ox1), and TFs (PtrWND6A, PtrWND6B, PtrMYB21, PtrMYB28, PtrMYB152, and PtrMYB52).

FIGURE 8

PtrWOX13A directly binds to the promoters of the poplar transcription factors and secondary cell wall biosynthesis pathway genes. (A) Schematic diagrams of the promoters showing the locations amplified by ChIP-PCR. (B) ChIP-PCR analysis of the promoter fragments enriched due to PtrWOX13A binding during immunoprecipitation; 30-day-old poplar plants overexpressing PtrWOX13A-Flag used for ChIP with anti-Flag antibodies. Input, Mock, and IP indicate the chromatin before immunoprecipitation (IP), IP with no antibodies, and IP with anti-FLAG antibodies, respectively. Those genes include lignin biosynthesis genes (PtrC4H1 and Ptr4CL5), fiber cell elongation genes (PtrXTH5 and PtrEXPA8), GA biosynthesis genes (PtrGA20ox4 and PtrGA3ox1), and TFs (PtrWND6A, PtrWND6B, and PtrMYB28).

FIGURE 9

Postulated regulatory model of the PtrWOX13A regulatory network.