Endoplasmic reticulum-localized UBC34 interaction with lignin repressors MYB221 and MYB156 regulates the transactivity of the transcription factors in Populus tomentosa

Abstract

Background: Regulation of lignin biosynthesis is known to occur at the level of transcription factors (TFs), of which R2R3-MYB family members have been proposed to play a central role via the AC cis-elements. Despite the important roles of TFs in lignin biosynthesis, the post-translational regulation of these TFs, particularly their ubiquitination regulation, has not been thoroughly explored.

Results: We describe the discovery of a Populus tomentosa E2 ubiquitin-conjugating enzyme 34 (PtoUBC34), which is involved in the post-translational regulation of transactivation activity of lignin-associated transcriptional repressors PtoMYB221 and PtoMYB156. PtoUBC34 is localized at the endoplasmic reticulum (ER) membrane where it interacts with transcriptional repressors PtoMYB221 and PtoMYB156. This specific interaction allows for the translocation of TFs PtoMYB221 and PtoMYB156 to the ER and reduces their repression activity in a PtoUBC34 abundance-dependent manner. By taking a molecular biology approach with quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we found that PtoUBC34 is expressed in all aboveground tissues of trees in P. tomentosa, and in particular, it is ubiquitous in all distinct differentiation stages across wood formation, including phloem differentiation, cambium maintaining, early and developing xylem differentiation, secondary cell wall thickening, and programmed cell death. Additionally, we discovered that PtoUBC34 is induced by treatment with sodium chloride and heat shock.

Conclusions: Our data suggest a possible mechanism by which lignin biosynthesis is regulated by ER-localized PtoUBC34 in poplar, probably through the ER-associated degradation (ERAD) of lignin-associated repressors PtoMYB221 and PtoMYB156.

Keywords: ER-associated degradation; Lignin biosynthesis; PtoMYB156; PtoMYB221; PtoUBC34; Transcriptional repressor.

Fig. 1

The specific interaction between PtoMYB221 and the N35 protein in a split-ubiquitin Y2H system. Bait protein PtoMYB221 fused to Cub was co-expressed in yeast NMY51 with prey proteins, NubI, NubG, or the NubG:N35 protein. NubI is a positive prey control with strong affinity between wild-type NubI and Cub, whereas NubG is a negative prey control with weak affinity between mutant NubG and Cub. Interaction of the bait was shown with positive control NubI and the test NubG:N35 fusion protein but not with the negative control NubG, as indicated by growth of the transformed strain on synthetic defined medium without Trp, Leu, and His and by the activity of β-Gal

Fig. 2

Characteristics of PtoUBC34. a. Phylogenetic analysis of PtoUBC34 and other ERAD-related UBC proteins in yeast (UBC6 and UBC7), humans (UBE2J1, UBE2J2, and UBE2G2), poplar, and Arabidopsis. The phylogenetic tree was established with full-length protein sequences by the neighbor-joining method. The bootstrap values out of 500 retrials are indicated at each branch. The scale (0.1) represents a 10% change in sequences. b. Schematic of PtoUBC34. The UBCc domain (blue) and a transmembrane domain (orange) are predicted in PtoUBC34 at the N-terminus and the C-terminus, respectively. c. Subcellular localizatin of PtoUBC34. PtoUBC34 was fused with YFP and co-transformed with the ER marker BiP:RFP [57] into P. tomentosa protoplasts. d. Multiple sequence alignment of yeast UBC6, human UBE2J2, Arabidopsis UBC34 and P. tomentosa UBC34. The conserved active Cys residue with red star, E3 interaction residues with black stars and Ub thioester intermediate interaction residues with blue stars are indicated. The transmembrane regions are shown in boxes. Bar in (c) = 10 μm

Fig. 3

PtoUBC34 is expressed ubiquitously and responds to stress factors. a. qRT-PCR analysis of PtoUBC34 expression in various poplar tissues, using Actin6 (Potri.001G309500), eIF-5A (Potri.018G107300), and UBQ (Potri.014G115100) genes as internal controls, according to Wang et al. [94]. The expression level of PtoUBC34 in leaves was set as 1. b. The expression levels of PtoUBC34 in mature leaves in response to heat shock treatment by qRT-PCR, using Actin6 (Potri.001G309500) and EF1-beta (Potri.009G018600) genes as internal controls, which were validated in an evaluation assay of eight reference genes during heat shock treatment in P. tomentosa (unpublished data). Plants were treated at 37 °C for 1 h (HS1), allowed to recover for 2 h at 24 °C (HSR), and then were subjected to 42 °C for 2.5 h (HS2). The fold-expression was normalized relative to the 24 °C control (CK). c. The expression levels of PtoUBC34 in mature leaves in response to 300 mM sodium chloride (NaCl) treatment were analyzed by qRT-PCR, using UBQ (Potri.014G115100) and TUB (Potri.003G126800) genes as internal controls, which were validated in an evaluation assay of eight reference genes during salt stress in P. tomentosa (unpublished data). The fold-expression was normalized relative to the non-NaCl-treatment control. Data are mean of three biological samples from three plants, respectively, with three technical replicates for each one. Error bar represents standard deviation of the three biological samples. Significance was tested with one-way ANOVA to evaluate the effect of heat shock and NaCl treatment on UBC34 expression. The different alphabets above the bar indicates statistically significant differences with p value < 0.05, while the same alphabet mean no significant differences. d. UBC34 from P. trichocarpa was expressed constitutively in distinct differentiation stages across wood formation. The expression data are derived from the ASPWOOD project (http://aspwood.popgenie.org/aspwood-v3.0/) [58]. Phloem, cambium, developing xylem, and mature xylem were collected from stems through longitudinal continuous sections using a cryo-microtome. The tissue type was characterized by examining the images of cross-sections during sampling. All of the samples were collected during the current growth year. Poplar CesA8-B, specifically expressed during SCW thickening, was used as a reference

Fig. 4

The Y2H demonstrated that full-length PtoUBC34 interacted with both the PtoMYB221 and PtoMYB156 protein in a split-ubiquitin Y2H system. Co-expression with NubI and NubG was used as positive and negative controls, respectively

Fig. 5

Protein co-localization in poplar mesophyll protoplasts. a and b. Protein co-localization of PtoUBC34 and PtoMYB221. c and d. Protein co-localization of PtoUBC34 and PtoMYB156. Both the PtoMYB221 and PtoMYB156 were fused with YFP and were co-transferred with the nuclear marker CBF1:RFP or RFP:PtoUBC34s into poplar protoplasts. The co-localization showed that TFs PtoMYB221 and PtoMY156 were translocated from the nucleus (a and c) to the ER (b and d) by PtoUBC34. Bar = 10 μm

Fig. 6

PtoUBC34 interacts with both PtoMYB221 and PtoMYB156 in vivo. a. BiFC in poplar protoplasts demonstrated that PtoUBC34 interacts with both the PtoMYB221 and PtoMYB156 in the ER. The BiFC signals co-localized with the marker Bip:RFP in the ER, suggesting that PtoUBC34 and each of PtoMYB221 and PtoMYB156 were co-localized in the same BiFC complex in the ER. Bar = 10 μm. b. Coimmunoprecipitation analysis showed an interaction of PtoUBC34s with both the PtoMYB221 and PtoMYB156. PtoUBC34s:Flag was co-expressed with each of MYB221:Myc and MYB156:Myc in poplar protoplasts. Protein extracts were incubated with anti-Flag coupled agarose. Immunoprecipitates (IP) and input proteins were analyzed by immunoblotting using anti-Flag and anti-Myc antibodies as indicated

Fig. 7

PtoMYB221 and PtoMYB156 are transcriptional repressors. a. Schematic representation of the constructs used for evaluation of the repression activity of PtoMYB221 and PtoMYB156 in dual LUC assay in poplar mesophyll protoplasts. The EAR-like motif repression domain of SUPERMAN (SUPRD) was used as a reference repressor [93]. b. Relative LUC activities after co-transformation with the reporter and effectors, where pUC18 was used as a control vector. Three independent experiments show similar results. Error bar represents standard deviation of three independent batches of P. tomentosa mesophyll protoplast transfections from one of the experiments. Significance was tested with one-way ANOVA to evaluate the roles of the effectors on the LUC expression. The different alphabets above the bar indicates statistically significant differences with p value < 0.05, while the same alphabet mean no significant differences

Fig. 8

PtoMYB221 targets to the AC cis-elements of monolignol pathway genes and its repression activity is reduced with the involvement of PtoUBC34. a. Schematic representation of the effector and reporter plasmids used for evaluation of the affinity of PtoMYB221 to AC cis-elements in a dual LUC assay. b. Synthetic AC elements tested in dual LUC assay. c. LUC activity assay driven by PtoMYB221 binding to the AC elements in poplar mesophyll protoplasts. AC-I, AC-II, AC-III, and AC-IV are the reporters containing three copies of the ACx elements fused to the CaMV35S promoter and LUC reporter gene. d. PtoUBC34 reduced the repression activity of PtoMYB221. 2.4 μg of the effector plasmids PtoMYB221, 600 ng of PtoUBC34 or PtoUBC34m, along with the reporter plasmid and internal control plasmid, were used for co-transfection. Three independent experiments show similar results. Error bar represents standard deviation of three independent batches of P. tomentosa mesophyll protoplast transfections from one of the experiments. Significance was tested with one-way ANOVA to evaluate the roles of the effectors on the LUC expression. The different alphabets above the bar indicates statistically significant differences with p value < 0.05, while the same alphabet mean no significant differences

Fig. 9

PtoUBC34 reduced the repression activity of PtoMYB221 in a dose-dependent manner. All LUC activities are expressed relative to values obtained after transformation with only the reporter plasmid, which was set at 100%. Three independent experiments show similar results. Error bar represents standard deviation of three independent batches of P. tomentosa mesophyll protoplast transfections from one of the experiments. Significance was tested with one-way ANOVA to evaluate the roles of the effectors on the expression level of LUC reporter gene. The different alphabets above the bar indicates statistically significant differences with p value < 0.05, while the same alphabet mean no significant differences

Fig. 10

Model of the roles of PtoUBC34 on the regulation of lignin biosynthesis. a. Schematic of cell with on-duty UBC34, and the gray part indicated PtoMYB221 absence in nucleus. b. Schematic of cell with off-duty UBC34, and the gray part indicated PtoMYB221 absence in ER compartments