Dimerization of PtrMYB074 and PtrWRKY19 mediates transcriptional activation of PtrbHLH186 for secondary xylem development in Populus trichocarpa

Abstract

Wood formation is controlled by transcriptional regulatory networks (TRNs) involving regulatory homeostasis determined by combinations of transcription factor (TF)-DNA and TF-TF interactions. Functions of TF-TF interactions in wood formation are still in the early stages of identification. PtrMYB074 is a woody dicot-specific TF in a TRN for wood formation in Populus trichocarpa. Here, using yeast two-hybrid and bimolecular fluorescence complementation, we conducted a genome-wide screening for PtrMYB074 interactors and identified 54 PtrMYB074-TF pairs. Of these pairs, 53 are novel. We focused on the PtrMYB074-PtrWRKY19 pair, the most highly expressed and xylem-specific interactor, and its direct transregulatory target, PtrbHLH186, the xylem-specific one of the pair's only two direct TF target genes. Using transient and CRISPR-mediated transgenesis in P. trichocarpa coupled with chromatin immunoprecipitation and electrophoretic mobility shift assays, we demonstrated that PtrMYB074 is recruited by PtrWRKY19 and that the PtrMYB074-PtrWRKY19 dimers are required to transactive PtrbHLH186. Overexpressing PtrbHLH186 in P. trichocarpa resulted in retarded plant growth, increased guaiacyl lignin, a higher proportion of smaller stem vessels and strong drought-tolerant phenotypes. Knowledge of the PtrMYB074-PtrWRKY19-PtrbHLH186 regulation may help design genetic controls of optimal growth and wood formation to maximize beneficial wood properties while minimizing negative effects on growth.

Keywords: Populus trichocarpa; protein-protein interaction; secondary xylem development; transcription factor; wood formation.

Fig. 1

PtrMYB074 Interacts with a Set of Transcription Factors. (a) Y2H demonstrated that PtrMYB074 interacts with 54 xylem‐abundant TFs. Each bait and prey pair was co‐expressed in yeast cells and selected on the SD/–Leu/–Trp/X‐α‐Gal/AbA (–LW/X/A), SD/–Leu/–Trp/–His/X‐α‐Gal/AbA (–LWH/X/A) and SD/–Leu/–Trp/–His/–Ade/X‐α‐Gal/AbA (–LWHA/X/A) medium. BD‐53/AD‐Lam were used as negative controls. BD‐53/AD‐T were used as positive controls. (b) Bimolecular fluorescence complementation (BiFC) demonstrated that PtrMYB074 interacts with PtrWRKY19 in vivo. PtrMYB074‐YFP^N and PtrWRKY19‐YFP^C (I) co‐expressed into Populus trichocarpa stem‐differentiating xylem protoplasts gave a positive BiFC signal for heterodimerization, which are co‐localized with H2A‐1:mCherry signal in the nucleus. PtrMYB021, an interacting protein of PtrMYB074 (Chen et al., 2019), was used as a positive control (II). As negative controls, the transformation of single TF‐YFP^N/C with YFP^C/N alone did not yield any YFP signals (III and IV). Green represents the YFP signals from protein interactions, red indicates the nuclear marker H2A‐1:mCherry, and yellow shows the merged signals from YFP and mCherry. Bar, 10 µm. YFP, yellow fluorescent protein.

Fig. 2

PtrWRKY19 Is Required for the Association of PtrMYB074 with the PtrbHLH186 Promoter. (a) A schematic diagram of the PtrbHLH186 loci. Triangles show the two primers used for the ChIP‐qPCR analysis. The wedge represents the W‐box in the PtrbHLH186 promoter. (b, e) Chromatin immunoprecipitation (ChIP) assays showing that PtrMYB074 and PtrWRKY19 associate with the promoter of the PtrbHLH186 gene. Wild‐type (WT), PtrWRKY19‐FLAG and PtrMYB074‐FLAG transgenic Populus trichocarpa plants were used for the ChIP assays with anti‐FLAG antibodies, and the precipitated DNA was quantified by qPCR. Enrichment of DNA was calculated as the ratio between PtrWRKY19‐FLAG and wild‐type or between PtrMYB074‐FLAG and wild‐type, normalized to that of the PtrActin gene. (c) Nucleotide sequences of the wild‐type W‐box and a mutated W‐box (mW‐box). Core sequences are shaded in black, and the mutated nucleotide is shaded in grey. (d) Electrophoretic mobility shift assay (EMSA) analysis of PtrWRKY19 binding to the W‐box in the PtrbHLH186 promoter. (f) PtrMYB074 alone failed to bind to the W‐box in the PtrbHLH186 promoter, and PtrWRKY19 is required for the association of PtrMYB074 with the PtrbHLH186 promoter. The arrow shows the shifted band representing the protein–DNA complex. PET101‐His was used as a negative control. The PtrbHLH186 promoter fragment was labelled with biotin. Fragments without biotin labelling were used as competitors. Wild‐type or mutated W‐box competitors were used in a molar excess of 50×, 100× or 200×. (g) ChIP assays at the promoter of PtrbHLH186 in WT and ptrwrky19 with anti‐IgG and anti‐PtrMYB074 antibodies, and the precipitated DNA was quantified by qPCR. Error bars in b, e and g indicate SE from three biological replicates, and the asterisk indicates significant differences between the control fragment (PtrActin) and the fragment containing a W‐box motif (*, P < 0.05, Student's t‐test).

Fig. 3

Co‐expression of PtrMYB074 and PtrWRKY19 Is Required for the Transcriptional Activation of PtrbHLH186. (a) Quantitative PCR detection of the transcript abundance of PtrbHLH186 in Populus trichocarpa stem‐differentiating xylem (SDX) protoplasts overexpressing GFP (control), PtrMYB074, PtrWRKY19 and PtrMYB074 together with PtrWRKY19 (PtrMYB074+PtrWRKY19). The control value was set as 1. Error bars indicate SE values of three biological replicates (three independent batches of SDX protoplast transfections). The asterisk indicates significant differences between GFP and the PtrMYB074‐PtrWRKY19 heterodimer (*, P < 0.05, Student's t‐test). GFP, green fluorescent protein. (b) The transcript abundance of PtrbHLH186 was detected using RT‐qPCR in xylem tissues of wild‐type, ptrmyb074 and ptrwrky19 plants. The control value was set as 1. Error bars indicate SE values of three biological replicates from independent pools of P. trichocarpa xylem tissues, and asterisks indicate significant differences between each mutant and wild‐type plants (**, P < 0.01, Student's t‐test).

Fig. 4

Effects of Overexpressing PtrbHLH186 on Populus trichocarpa Growth. (a) The growth phenotypes of 4‐month‐old OE‐PtrbHLH186 (L1, L2, L3 and L4) and wild‐type (WT) plants. Bar, 10 cm. (b) The expression levels of the PtrbHLH186 transgene in stem‐differentiating xylem tissues of four PtrbHLH186 transgenic lines (L1, L2, L3 and L4). Error bars represent SE values from three biological replicates with five P. trichocarpa plants for each genotype in each replicate. The asterisk indicates significant differences between each line of the transgenics and wild‐type plants by Student's t‐test (*, P < 0.05; **, P < 0.01). (c–e) Statistical analysis of height, internode number and stem basal diameter of wild‐type and OE‐PtrbHLH186 (L1, L2, L3 and L4) transgenic plants. Error bars represent SE of three independent experiments with 5 P. trichocarpa plants for each genotype in each replicate. The asterisk indicates significant differences between each line of the transgenics and wild‐type plants by Student's t‐test (**, P < 0.01). OE, overexpressing.

Fig. 5

Expression Levels of Monolignol Genes, and Model Predicted and Measured G and S subunits in Lignin of OE‐PtrbHLH186‐L4 and Wild‐type Populus trichocarpa. (a) The stem‐differentiating xylem (SDX) of wild‐type and OE‐PtrbHLH186‐L4 transgenic plants was used to perform the quantitative PCR. Error bars indicate SE values of three biological replicates from independent pools of P. trichocarpa SDX tissues (**, P < 0.01; *, P < 0.05, Student's t‐test). (b, c) Multi‐omics integration predicts increased G subunits and reduced S subunits in lignin of OE‐PtrbHLH186‐L4. The transcript abundance of monolignol biosynthetic genes in OE‐PtrbHLH186‐L4 and wild‐type P. trichocarpa was used as the input for a multi‐omics predictive model of lignin biosynthesis (Wang et al., 2018) to estimate the corresponding levels of G monolignol (b) and S monolignol (c) subunits in lignin. Error bars represent 1 SE of three technical replicates. (d) The scatterplot depicts the linear regression of lignin subunits predicted by the multi‐omics integrative model (Wang et al., 2018) and measured lignin subunits from wood chemistry analysis of OE‐PtrbHLH186‐L4 and wild‐type P. trichocarpa (Table 1). The predicted and measured values have an R ² of 0.89. OE, overexpressing.

Fig. 6

Overexpressing PtrbHLH186 Affects the Size and Number of Vessels in Xylem Tissue and Improves Drought Tolerance of Populus trichocarpa. (a) Scanning electron micrographs of wild‐type (WT) and OE‐PtrbHLH186‐L4 (L4) with the 10th internode imaged at ×500 (left) and ×1500 (right) magnification. Bar, 20 µm. (b) Stem cross sections and magnified images of 4‐month‐old OE‐PtrbHLH186‐L4 and wild‐type plants with the 10th internode (IN). The cross sections were stained with Safranin O and Fast Green. Bar, 100 µm. (c) Stem cross sections of OE‐PtrbHLH186‐L4 and wild‐type plants showing the size and number of vessel cells in xylem tissues. Vessel cells are marked with green. Bar, 100 µm. (d) Magnified images of tangential longitudinal sections stained with toluidine blue. Bar, 100 µm. F, xylem fibre; V, vessel element. (e–i) Statistical analyses of the number of fibres and vessels per cross‐sectional area (mm²; e), mean lumen area of individual vessel (μm²; f), length of fibres and vessels (μm; g), area of vessels (μm²) per cross‐sectional area (mm²; h) and cell wall thickness of fibres and vessels (μm; i) within the 10th internode. Error bars represent SE values of three independent replicates with at least 200 vessel cells and 3000 fibre cells for each genotype in each replicate, and asterisks indicate significant differences between the transgenic and wild‐type plants by Student's t‐test (**, P < 0.01). (j) Statistical analysis of survival rates after drought treatment and recovery (rehydrated for 3 d). Error bars represent SE values from three biological replicates. Asterisks indicate significant differences between the transgenics and wild‐type plants (**, P < 0.01, Student's t‐test). OE, overexpressing.