A Molecular Blueprint of Lignin Repression

TL;DR: This work provides a comprehensive overview of the molecular factors that negatively impact on the lignification process at both the transcriptional and post-transcriptional levels.

About: This article is published in Trends in Plant Science.The article was published on 2019-11-01 and is currently open access. It has received 20 citations till now. The article focuses on the topics: Lignocellulosic biomass.

Summary

Review

• The Mediator complex adds another level of transcription regulation to the several transcription factors that are known to repress.
• The need to tailor the lignocellulosic biomass for more efficient biofuel production or for improved plant digestibility has fostered considerable advances in their understanding of the lignin biosynthetic pathway and its regulation.
• The authors provide a comprehensive overview of the molecular factors that negatively impact on the lignification process at both the transcriptional and post-transcriptional levels.
• Understanding the interactions between genes, non-coding RNAs, and proteins opens new avenues towards understanding secondary cell wall formation.

Transcriptional Repression of Lignin Biosynthesis

• Negative regulation of lignin biosynthesis is achieved through diverse mechanisms ranging from DNA accessibility to targeted proteolysis.
• The process, the timing, and the location of differentiation are under stringent genetic regulation.
• Usually TFs, also known as Heterodimer.
• An RNA that is not translated into protein, also known as Non-coding RNA.

NAC TFs, the Two Sides of SCW Regulation

• Members of the NAC family act as first- and second-level master switches in the regulation of a battery of downstream TFs and SCW biosynthetic genes [15–18].
• VND-INTERACTING 2 (VNI2) is a transcriptional repressor reported to regulate the timing and spatial regulation of xylem cell development [21].
• The SCW activator NST2 is negatively transcriptionally regulated by WRKY12 , which binds to the W-box cis-element in theNST2 promoter region (Table 1) [25].
• An intron-retained (IR) splice variant PtrVND6C1IR negatively regulates the expression of PtrMYB021 (a poplar ortholog of AtMYB46) by forming heterodimers with the full-size PtrVND6s, suppressing their positive transcriptional activity .
• In addition, PtrVND6-C1IR downregulates the expression of five full-size PtrVND6s.

Key Figure

• PtrhAT PtrMYB021 Transcriptional complex LAC AtVNDs AtVND7 AtVNI2 AtXND1 Active PtrAldOMT2 P Ser 123 Ser125 Inactive PtrAldOMT2 LTF1 Phosphorylated LTF1 U EgH1.3 TrendsinPlantScience.
• In this review the authors describe alternatively spliced proteins regulating the expression of closely related coding genes.

R2R3 MYBs, the Gatekeepers of SCW Formation and Lignification

• Some members of the R2R3-MYB TF family positively regulate gene expression of phenylpropanoid and lignin biosynthetic genes containing AC-rich cis-elements in their promoters [30], such as the 7 bp sequence ACC(A/T)A(A/C)(T/C), termed the secondary wall MYB-responsive element (SMRE) [31,32].
• The importance of MYBs as repressors of phenylpropanoid metabolism has been highlighted in a recent review [33].
• AtMYB4 belongs to subgroup 4 and, as the other proteins from this subgroup (AtMYB3, AtMYB7, and AtMYB32), contains an EARlike repression motif in its C-terminus [36].
• AtMYB4 is downregulated in thale cress ectopic lignification de-etiolated 3, pom-pom 1, and ectopic lignification 1 mutants [38], suggesting that it could negatively regulate lignin biosynthesis.
• Notably, PtrEPSP-TF harbors an additional N-terminal HTH DNA-binding motif that partially targets this protein to the nucleus, where it acts as a transcriptional repressor of its direct target PtrhAT, a hAT transposase family gene.

KNOX, BELL, and Homeodomain: from Cell Division to Fiber SCW Thickening

• Some members of the THREE AMINO ACID LOOP EXTENSION (TALE) family of homeodomain (HD) proteins may play a role in the repression of lignin biosynthesis .
• The cooperative heterodimer becomes completely contained in the nucleus, and the expression of the target genes is dramatically reduced relative to individual BELL or KNOX proteins [22,71].
• The heterodimer KNAT7–BLH6 negatively regulates the commitment to SCW formation in interfascicular fibers of thale cress through repression of REVOLUTA , which encodes a HD-leucine zipper TF binding to the sequence GTAATNATTAC [65,72].
• Indeed, the athb15 mutant showed increased xylan and lignin contents in the pith as well as higher expression of SCW genes [81].
• Of note, KNOX are also part of the transcriptional network regulating the formation of tension wood in poplar [85] that is characterized by the presence of a thick, weakly lignified, cellulose-rich gelatinous layer.

Mediator, a Molecular Hub Coordinating Lignin Biosynthesis with Plant Growth

• The ’mediator of RNA polymerase II transcription’, or Mediator complex (MED), is essential to transduce signals (both positively and negatively regulating gene expression) to the transcription machinery via direct interactions with specific TFs [86].
• Among the 27 MED subunits identified in thale cress [87], several negatively regulate the phenylpropanoid and monolignol biosynthetic pathways, contributing to the homeostasis of this family of secondary metabolites.
• The lignin monomeric composition is drastically modified in the triple mutant, consisting almost exclusively of H-lignin subunits (95% vs <2% in the wild type), suggesting that MED5a and MED5b are likely to have other functions [90].
• Dolan and colleagues [91] have also demonstrated that the MED5b phenotype requires functional MED2, MED16, and MED23, which probably physically and functionally interact with MED5, as do their homologs in humans [92].

Post-Transcriptional Repression of Monolignol Biosynthesis and Lignin Polymerization

• In addition to the numerous mechanisms of transcriptional regulation that land plants have established to repress monolignol biosynthesis and hence lignification in different tissues and developmental stages, additional post-transcriptional mechanisms have been observed.
• Post-transcriptional modifications typically affect a restricted number of transcripts/proteins, allowing precise control of the output of a metabolic pathway such as lignin biosynthesis.

Non-Coding RNAs, Emerging Regulators for Genetic Control of Lignin Deposition

• MicroRNAs are small non-coding RNAs that post-transcriptionally regulate many aspects of plant development.
• Their expression is developmentally regulated and/or under the control of external stimuli such as abiotic stress or nutrient availability [93,94].
• Overexpression of ptr-miR397a significantly reduces the expression of 17 of the 34 LAC found in poplar differentiating xylem, the global LAC activity of this tissue, and the lignin content of the whole plant [26].
• Similarly, 18 conserved miRNAs targeting 80 genes were found in hemp, where they may have similar functions to flax miRNAs [98].
• These lncRNAs may be directly functional or serve as precursors for miRNA sequences such asmiR397 [101], and provide a further level of complexity in the regulation of lignin biosynthesis.

Protein Ubiquitination: the Signaling Wave to the Grave

• PAL catalyzes the rate-limiting step of the phenylpropanoid pathway and thus constitutes an ideal target for regulating the flux of derived secondary metabolites.
• Thale cress KFB01, KFB20, KFB39, and KFB50 physically interact with the four PAL isozymes, thereby regulating the biosynthesis of phenylpropanoids during plant development and in response to environmental stimuli [27,103].
• The hemp ortholog of KFB39 is upregulated in mature bast fibers, suggesting a role for KFBs in the hypolignification of this cell type [83].

Switching On/Off Enzymatic Activity with Phosphorylation

• Phosphorylation is a widespread post-translational modification which may impact on the lignification process.
• Monophosphorylation of PtrAldOMT2 (that catalyzes the methylation of 5-hydroxyconiferaldehyde to sinapaldehyde) at either Ser123 or Ser125 inhibits its activity [105], in line with the observation that the pool of monolignol biosynthetic enzymes is usually not phosphorylated in vivo [106].
• The biological significance of this switch remains unknown.
• Alternatively, phosphorylation may also constitute a signal for protein degradation through proteasome activity.
• By screening TFs binding to the poplar 4CL promoter, Gui and colleagues identified a lignin biosynthesis-associated factor, LTF1, that represses several genes from this pathway (PAL2, C4H1, C3H2, 4CL1, CAld5H, COMT2, and CCoAOMT1) and decreases lignin content in overexpressing lines [107].

Concluding Remarks and Future Perspectives

• Further advances in synthetic and molecular biology combine with their growing knowledge about the molecular factors (mainly genes and proteins) driving SCW formation in various tissues and plant species to overcome the possible growth penalty of constitutive overexpression of genes repressing lignification (see Outstanding Questions).
• Similarly, the dwarf thale cress ccr1 mutant was rescued by driving the expression of CCR1 in metaxylem and protoxylem vessels through a proSNBE promoter transcriptionally activated by VND6 and VND7 [109].
• Targeted lignin biosynthesis repression may thus be achieved through temporal and/or spatial restriction of the activity of a selected gene using suitable promoters.
• Omics-based predictive analysis of variables determining wood quality following targeted gene downregulation [110] constitutes a valuable tool to optimize strategies.
• DNA methylation contributes to the regulation of cotton fiber development and can modulate the production of reactive oxygen species or the biosynthesis of lipids, flavonoids, and ascorbate [111].

Acknowledgments

• G. Guerriero acknowledges support from the Fonds National de la Recherche, Luxembourg (grant number C16/SR/ 11289002).
• J. Grima-Pettenati acknowledges support from the CNRS, the Université Paul Sabatier Toulouse III, and the Laboratoire d’Excellence TULIP (ANR-10-LABX-41; ANR-11- IDEX0002-02).

Figures

Figure 2. PAL as a Case Study ofMultipleMechanisms Regulating Gene Expression and Protein Abundance. (A) PAL is directly repressed through binding of various TALE family transcription factors (TFs; e.g., BREVIPEDICELLUS) to the DNA knotted 1 (KN-1) motif, as well as by binding of subgroup 4 R2R3 MYBs (e.g., MYB4) to the AATAGTT motif. (B) PAL is indirectly regulated through BLH6–KNAT7-mediated repression of the HD-Zip III TF REVOLUTA. HB15 represses the expression of the master switches SND1 and NST2, which are activators of PAL expression. (C) Kelch F-box proteins ubiquitinate PAL to target it to the 26S proteasome for proteolysis. Other PAL regulatory pathways, including metabolic feedback repression (caffeic acid, cinnamic acid), environmental factors, and physical interactions with other phenylpropanoid enzymes (C4H) are beyond the scope of this figure but are reviewed in [44]. Abbreviations: BLH, BEL1-like homeodomain; HB, homeobox; KFB, kelch F-box; KNAT, knotted1-like TALE homeodomain; NST, NAC secondary wall thickening promoting factor; PAL, phenylalanine ammonia lyase; SND, secondary wall-associated NAC domain protein; Ub ubiquitin. This figure was created using BioRender (https://biorender.com/).

Table 1. Binding Sites of Proteins Involved in the Repression of Monolignol Biosynthesis

Figure 1. The repressive activity of EgMYB1 increases when it is associated with EgH1.3. Some factors are devoted to specifically preventing monolignol biosynthesis (blue boxes), whereas others repress secondary cell wall (SCW) formation more widely (violet boxes). Proteins repressing SCW formation are essentially involved in the spatiotemporal regulation of specific tissue development, such as preventing SCW deposition in pith (AtWRKY12, AtHB15) or restricting the timing and localization of xylem cell development (AtVNI2). Abbreviations: 4CL, 4-coumarate-CoA ligase; AldOMT, 5-hydroxyconiferaldehyde O-methyltransferase; ARK, arborknox; BLH, BEL1-like homeodomain; BP, brevipedicellus; C4H, cinnamate-4-hydroxylase; DRIF, divaricata and radialis interacting factor; H1.3, linker histone variant; hAT, hobo activator Tam3 transposase; HB, homeobox; KFB Kelch F-box; KNAT, knotted1-like TALE homeodomain; LAC, laccase; LTF, lignin biosynthesisassociated transcription factor; MED, Mediator; NST, NAC secondary wall thickening promoting factor; OFP, ovate family protein; P, phosphorylation; PAL, phenylalanine ammonia lyase; PCN, popcorona; SND, secondary wall-associated NAC domain protein; U, ubiquitin; VND vascular-related NAC domain; VNI, VND interacting; XND, xylem NAC domain. This figure was created using BioRender (https://biorender.com/).