Characterization of a High Hierarchical Regulator, PtrGATA12, Functioning in Differentially Regulating Secondary Wall Component Biosynthesis in Populus trichocarpa

Abstract

In plants, GATA transcription factors (TFs) have been reported to play vital roles in to a wide range of biological processes. To date, there is still no report about the involvement and functions of woody plant GATA TFs in wood formation. In this study, we described the functional characterization of a Populus trichocarpa GATA TF, PtrGATA12, which encodes a nuclear-localized transcriptional activator predominantly expressing in developing xylem tissues. Overexpression of PtrGATA12 not only inhibited growths of most phenotypic traits and biomass accumulation, but also altered the expressions of some master TFs and pathway genes involved in secondary cell wall (SCW) and programmed cell death, leading to alternated SCW components and breaking forces of stems of transgenic lines. The significant changes occurred in the contents of hemicellulose and lignin and SCW thicknesses of fiber and vessel that increased by 13.5 and 10.8%, and 20.83 and 11.83%, respectively. Furthermore, PtrGATA12 bound directly to the promoters of a battery of TFs and pathway genes and activated them; the binding sites include two cis-acting elements that were specifically enriched in their promoter regions. Taken together, our results suggest PtrGATA12, as a higher hierarchical TF on the top of PtrWND6A, PtrWND6B, PtrMYB152, and PtrMYB21, exert a coordinated regulation of SCW components biosynthesis pathways through directly and indirectly controlling master TFs, middle-level TFs, and further downstream pathway genes of the currently known hierarchical transcription network that governs SCW formation.

Keywords: Populus trichocarpa; PtrGATA12; biological characterization; coordinated regulation; hierarchical regulator; secondary cell wall.

Figure 1

Phylogenetic analysis and protein sequence alignment of PtrGATA12. (A) Phylogenetic analysis of PtrGATA12 with other GATA proteins in Populus trichocarpa and Arabidopsis thaliana. PtrGATA12 proteins were shown in a red rectangle. The tree showed four major phylogenetic subfamilies (subfamilies I to IV) indicated with different colored backgrounds. (B) AtGATA12 protein (AT5G25830) of A. thaliana was aligned with PtrGATA12 proteins from P. trichocarpa (Potri.001G188500, Potri.006G237700, Potri.018G044900, and Potri.T158300). The conserved CLBC, TPQWR, and LCNACG in GATA motif are indicated by red box, blue box, and brown box, respectively; NLS, the putative domains for nuclear localization signals; the boxed region indicates the putative activation domain.

Figure 2

Tissue-specific expression patterns, subcellular localization and transcriptional activity of PtrGATA12. (A) The tissue-specific expression patterns of Potri.001G188500 gene as determined by quantitative RT-PCR (qRT-PCR) analysis. (B) The tissue-specific expression patterns of Potri.006G237700 gene as determined by qRT-PCR analysis. (C) The tissue-specific expression patterns of Potri.018G044900 gene as determined by qRT-PCR analysis. (D) The tissue-specific expression patterns of Potri.T158300 gene as determined by qRT-PCR analysis. PtrActin was used as a control. Error bars represent the SD of three biological replicates. (E) Subcellular localization of PtrGATA12. Confocal images manifested the localization of PtrGATA12-green fluorescent proteins (GFP) in the nuclei of onion epidermal cells. DAPI, a nuclear staining dye; Merged: the merged images of bright-field, GFP, and DAPI staining. Arrows indicate cells located in the epidermis of onions. (F) Transcriptional activation analysis of PtrGATA12 fused with the GAL4 DNA binding domain (GAL4DB) in yeast shows its potential to activate the expression of the His-3 and X-α-Gal reporter genes.

Figure 3

Identification and transgenic lines of PtrGATA12 in P. trichocarpa. (A) PCR detection of PtrGATA12 transgenic lines. M, DNA marker DL5000; 1, positive control; 2–8, PCR products of PtrGATA12 transgenic lines; WT, wild-type P. trichocarpa. (B) qRT-PCR detection of PtrGATA12 transgenic lines. PtrActin was used as a control. Each error bar represents the SD of three biological replicates. Asterisks indicate levels of significance (Dunnett’s test; *, 0.01 < p < 0.05, **, p < 0.01). (C) Three-month-old wild-type (WT) and PtrGATA12 transgenic lines (OE-1, OE-3, and OE-6). Scale bars = 10 cm.

Figure 4

Effect of PtrGATA12 overexpression on growth-related traits in P. trichocarpa. (A–H) Represent the leaf lengths, leaf widths, average lengths, plant heights, fresh weights, dry weights internode base diameters, and breaking forces of 90 days old WT and PtrGATA12 transgenic lines (OE-1, OE-3, and OE-6), respectively. Each error bar represents the SD of three biological replicates. Asterisks indicate levels of significance (Dunnett’s test; **, p < 0.01).

Figure 5

Effect of PtrGATA12 overexpression on the secondary wall thickness of stems in P. trichocarpa. (A–H) The scanning electron microscope of cross stem sections of 90 days old wild-type (A–D) and PtrGATA12 transgenic lines (E–H). Scale bars = 10 μm. (C,G) The scanning electron microscope of fiber walls in wild-type and PtrGATA12 transgenic lines. (D,H) The scanning electron microscope of vessel walls in wild-type and PtrGATA12 transgenic lines. (I) The fiber wall thickness of cross stem sections in WT and PtrGATA12 transgenic lines (OE-1, OE-3, and OE-6). (J) The vessel wall thickness of cross stem sections in WT and PtrGATA12 transgenic lines. Error bars represent SD of three biological replicates. Asterisks indicate levels of significance (Dunnett’s test; *, 0.01 < p < 0.05, **, p < 0.01).

Figure 6

Effects of PtrGATA12 overexpression on components of secondary cell wall of stems in P. trichocarpa. (A,D) Phloroglucinol-HCl staining (red color) stem sections of 90 days old wild-type (A) and PtrGATA12 transgenic lines (D). (B,E) Calcofluor white staining (blue color) stem sections of wild-type (B) and PtrGATA12 transgenic lines (E). (C,F) Monoclonal antibody LM10 (green color) stem sections of wild-type (C) and PtrGATA12 transgenic lines (F). Scale bars = 100 μm. (G) The lignin content of stems in WT and PtrGATA12 transgenic lines (OE-1, OE-3, and OE-6). (H) The cellulose content of stems in WT and PtrGATA12 transgenic lines. (I) The hemicellulose content of stems in WT and PtrGATA12 transgenic lines. Error bars represent SD of three biological replicates. Asterisks indicate levels of significance (Dunnett’s test; **, p < 0.01).

Figure 7

Expression analysis of wood formation pathway genes and their regulatory TFs in 90 days old wild-type and PtrGATA12 transgenic lines. Control represents the normalized expression level (namely 1 in this case) of each wood formation gene examined in wild-type plants. Error bars represent SD of three biological replicates. Asterisks indicate levels of significance (Dunnett’s test; **, p < 0.01).

Figure 8

Activation or repression of the promoters of poplar transcription factors and secondary cell wall (SCW) pathway genes by PtrGATA12. (A,B) Diagrams of the effector and reporter constructs. (C) The expression of the β-glucuronidase (GUS) under the control of those promoters by PtrGATA12. GUS activity in tobacco leaves transfected with the reporter construct alone was used as a control and was set to 1. Error bars represent SD of three biological replicates. Asterisks indicate levels of significance of differential expression (Dunnett’s test; *, 0.01 < p < 0.05, **, p < 0.01).

Figure 9

Yeast one-hybrid assay of PtrGATA12 binding to cis-acting elements. The pGADT7-rec2-p53/pHIS2-p53 and pGADT7-rec2-PtrGATA12/pHIS2-p53 were used as the positive and negative control, respectively. 10⁻¹, 10⁻², and 10⁻³ represent solution dilution ratio of transformed Y187 yeast cells.