Enzyme Complexes of Ptr4CL and PtrHCT Modulate Co-enzyme A Ligation of Hydroxycinnamic Acids for Monolignol Biosynthesis in Populus trichocarpa

Abstract

Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. In Populus trichocarpa, two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation of PtrHCTs reduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenic P. trichocarpa. The Ptr4CL/PtrHCT interactions were then validated in vivo using biomolecular fluorescence complementation (BiFC) and protein pull-down assays in P. trichocarpa SDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation in P. trichocarpa.

Keywords: BiFC; Populus trichocarpa; metabolic flux; monolignol biosynthesis; protein interaction; wood formation.

FIGURE 1

A section of the proposed monolignol biosynthetic pathway in Populus spp. The H, C, G, and S refer to the type of subunits in lignin, 4-hydroxyphenyl, catechyl, guaiacyl, and syringyl, respectively. Reaction A indicates 4CL activity for the formation of 4-coumaroyl-CoA from 4-coumaric acid, and reaction B indicates 4CL activity for the formation of caffeoyl-CoA from caffeic acid. C4H, cinnamate 4-hydroxylase; C3H, p-coumarate 3-hydroxylase; C3′H, p-coumaroyl shikimate 3′-hydroxylase; 4CL, 4-coumarate CoA ligase; HCT, 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferase; CSE, caffeoyl shikimate esterase. Enzymes in parenthesis indicate the distinguished reactions for the specific hydroxylation of monolignols.

FIGURE 2

Transcript abundance of SDX-specific Ptr4CLs and their activities in PtrHCT1 or PtrHCT6 RNAi-downregulated transgenic lines. (A) The CoA ligation activities toward 4-coumaric acid (Reaction A) and caffeic acid (Reaction B) using SDX extracts of WT, PtrHCT1, and PtrHCT6 RNAi-downregulated transgenic lines. The activities of Ptr4CL were measured using SDX extracts at 37°C for 30 min with 50 μM substrate (final concentration) in the assay solution [50 mM Tris–HCl buffer (pH 7.5), 2 mM MgCl₂, 2 mM ATP, and 0.2 mM CoA]. (B) Transcript abundance of Ptr4CL3 (in gray) and Ptr4CL5 (in black) in WT, PtrHCT1, and PtrHCT6 RNAi-downregulated transgenic lines. RPM, reads per million. Error bars represent the mean ± standard error (SE) from three biological replicates (n = 3). Statistical testing was performed using Student’s t-test (^∗p < 0.05; ^∗∗p < 0.01).

FIGURE 3

Protein-protein interactions between Ptr4CLs and PtrHCTs were detected by reciprocal bimolecular fluorescence complementation (BiFC). (A) Ptr4CL3-YFP^N was cotransformed with PtrHCT1-YFP^C. (C) Ptr4CL3-YFP^N was cotransformed with PtrHCT6-YFP^C. (E) Ptr4CL5-YFP^N was cotransformed with PtrHCT1-YFP^C. (G) Ptr4CL5-YFP^N was cotransformed with PtrHCT6-YFP^C. (B,D,F,H) Reciprocal BiFC assays of (A,C,E,G). (I–L) Negative controls of BiFC assays. The Gus-YFP^C were cotransformed with PtrHCT1-YFP^N, PtrHCT6-YFP^N (E), or Ptr4CL3-YFP^N, Ptr4CL5-YFP^N (F), as the negative controls.

FIGURE 4

Protein–protein interactions between Ptr4CLs and PtrHCTs were detected on western blots after pull-downs using SDX total protein extracts. SDX total protein extracts were independently incubated with recombinant proteins of Ptr4CL3, Ptr4CL5, PtrHCT1, and PtrHCT6 with a C-terminal 6 × His tag. The protein mixtures were affinity purified and analyzed by western blotting using anti-Ptr4CL3 (A) and anti-Ptr4CL5 (B) antibodies to identify interacting proteins. Recombinant proteins of Ptr4CL3, Ptr4CL5, PtrHCT1, PtrHCT6, and Gus were detected using His antibody (C). WB, western blot. Refer to Supplementary Figure 6 for the uncropped western blots.

FIGURE 5

CoA ligation activity with 4-coumaric acid (Reaction A) and caffeic acid (Reaction B) of recombinant Ptr4CL3 and Ptr4CL5 are affected by the presence of PtrHCT1 or PtrHCT6. (A) Reaction A of Ptr4CL3 with PtrHCTs. (B) Reaction B of Ptr4CL3 with PtrHCTs. (C) Reaction A of Ptr4CL5 with PtrHCTs. (D) Reaction B of Ptr4CL5 with PtrHCTs. Ptr4CLs were fixed at 10 nM, and PtrHCT concentration was fixed at 40 nM. Substrate concentration was fixed at 50 μM. Error bars represent one SE of three technical replicates. Statistical testing was performed using the Student’s t-test (**p < 0.01).

FIGURE 6

Impact of Ptr4CL-PtrHCT complex formation on the CoA ligation of 4-coumaric acid (Reaction A) or caffeic acid (Reaction B). (A) Reaction A of Ptr4CL3 with increasing PtrHCT1 concentration. (B) Reaction A of Ptr4CL3 with increasing PtrHCT1 concentration. (C) Reaction A of Ptr4CL5 with increasing PtrHCT1 concentration. (D) Reaction A of Ptr4CL5 with increasing PtrHCT6 concentration. (E) Reaction B of Ptr4CL3 with increasing PtrHCT1 concentration. (F) Reaction B of Ptr4CL3 with increasing PtrHCT1 concentration. (G) Reaction B of Ptr4CL5 with increasing PtrHCT1 concentration. (H) Reaction B of Ptr4CL5 with increasing PtrHCT6 concentration. The concentration of Ptr4CL3 or Ptr4CL5 was fixed as 10 nM. Dashed lines indicate the baseline of activity of each Ptr4CL. Error bars represent SE of three replicates.

FIGURE 7

Numerical analysis of CoA ligation activity for 4-coumaric acid (Reaction A) or caffeic acid (Reaction B) in model optimization process. (A) Ptr4CL3 to PtrHCT1 ratio for Reaction A. (B) Ptr4CL3 to PtrHCT6 ratio for Reaction A. (C) Ptr4CL5 to PtrHCT1 ratio for Reaction A. (D) Ptr4CL5 to PtrHCT6 ratio for Reaction A. (E) Ptr4CL3 to PtrHCT1 ratio for Reaction B. (F) Ptr4CL3 to PtrHCT6 ratio for Reaction B. (G) Ptr4CL5 to PtrHCT1 ratio for Reaction B. (H) Ptr4CL5 to PtrHCT6 ratio for Reaction B. RMSE, root mean squared error; BIC, Bayesian information criterion.

FIGURE 8

Detection of Ptr4CL-PtrHCT complexes in SDX extracts and recombinant proteins using chemical cross-linking by DSP. (A) Cross-linking of recombinant protein Ptr4CLs and PtrHCTs and detection by Ptr4CL3 specific antibody. Lane 1: Non-cross-linked recombinant Ptr4CL3 and PtrHCT1. Lane 2: Cross-linked recombinant Ptr4CL3 and PtrHCT1. Lane 3: Non-cross-linked recombinant Ptr4CL3 and PtrHCT6. Lane 4: Cross-linked recombinant Ptr4CL3 and PtrHCT6. (B) Cross-linking of recombinant protein Ptr4CLs and PtrHCTs and detection by PtrHCT1 antibody. Lane 1: Non-cross-linked recombinant Ptr4CL3 and PtrHCT1. Lane 2: Cross-linked recombinant Ptr4CL3 and PtrHCT1. Lane 3: Non-cross-linked recombinant Ptr4CL5 and PtrHCT1. Lane 4: Cross-linked recombinant Ptr4CL5 and PtrHCT1. (C) Detection of Ptr4CL-PtrHCT complexes in DSP cross-linked SDX extracts using PtrHCT1 specific antibody. (D) Detection of Ptr4CL-PtrHCT complex in DSP cross-linked SDX extract using PtrHCT6 specific antibody. WB, western blot. Refer to Supplementary Figure 6 for the uncropped western blots.