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Journal ArticleDOI

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 (3 min read)

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).

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Journal ArticleDOI
TL;DR: Overexpression of VlbZIP30 improves drought tolerance, characterized by a reduction in the water loss rate, maintenance of an effective photosynthesis rate, and increased lignin content in leaves under drought conditions.
Abstract: Drought stress severely affects grapevine quality and yield, and recent reports have revealed that lignin plays an important role in protection from drought stress. Since little is known about lignin-mediated drought resistance in grapevine, we investigated its significance. Herein, we show that VlbZIP30 mediates drought resistance by activating the expression of lignin biosynthetic genes and increasing lignin deposition. Transgenic grapevine plants overexpressing VlbZIP30 exhibited lignin deposition (mainly G and S monomers) in the stem secondary xylem under control conditions, which resulted from the upregulated expression of VvPRX4 and VvPRX72. Overexpression of VlbZIP30 improves drought tolerance, characterized by a reduction in the water loss rate, maintenance of an effective photosynthesis rate, and increased lignin content (mainly G monomer) in leaves under drought conditions. Electrophoretic mobility shift assay, luciferase reporter assays, and chromatin immunoprecipitation-qPCR assays indicated that VlbZIP30 directly binds to the G-box cis-element in the promoters of lignin biosynthetic (VvPRX N1) and drought-responsive (VvNAC17) genes to regulate their expression. In summary, we report a novel VlbZIP30-mediated mechanism linking lignification and drought tolerance in grapevine. The results of this study may be of value for the development of molecular breeding strategies to produce drought-resistant fruit crops.

43 citations

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Abstract: Lodging is a major problem limiting maize yield worldwide. However, the mechanisms of lodging resistance remain incompletely understood for maize. Here, we evaluated 443 maize accessions for lodging resistance in the field. Five lodging-resistant accessions and five lodging-sensitive accessions were selected for further research. The leaf number, plant height, stem diameter, and rind penetrometer resistance were similar between lodging-resistant and -sensitive inbred lines. The average thickness of sclerenchymatous hypodermis layer was thicker and the vascular area was larger in the lodging-resistant lines compared with lodging-sensitive lines. Although total lignin content in stem tissue did not significantly differ between lodging-resistant and -sensitive lines, phloroglucinol staining revealed that the lignin content of the cell wall in the stem cortex and in the stem vascular tissue near the cortex was higher in the lodging-resistant lines than in the lodging-sensitive lines. Analysis of strand-specific RNA-seq transcriptome showed that a total of 793 genes were up-regulated and 713 genes were down-regulated in lodging-resistant lines relative to lodging-sensitive lines. The up-regulated genes in lodging-resistant lines were enriched in cell wall biogenesis. These results indicated that modification of cell wall biosynthesis would contribute to lodging resistance of maize.

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TL;DR: The results provide evidence that both OsWRKY36 andOsWRKY102 are associated with repression of rice lignification, and relative abundances of guaiacyl and p-coumarate units were slightly higher and lower, respectively, in the WRKY mutant lignins compared with those in the wild-type lign ins.

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References
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Journal ArticleDOI
TL;DR: The results indicate conservation among MYB transcription factors in terms of their interaction with SAD2, and suggest that the Asp → Asn mutation may be used to engineer transcription factors.
Abstract: Sub-group 4 R2R3-type MYB transcription factors, including MYB3, MYB4, MYB7 and MYB32, act as repressors in phenylpropanoid metabolism. These proteins contain the conserved MYB domain and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) repression domain. Additionally, MYB4, MYB7 and MYB32 possess a putative zinc-finger domain and a conserved GY/FDFLGL motif in their C-termini. The protein 'sensitive to ABA and drought 2' (SAD2) recognizes the nuclear pore complex, which then transports the SAD2-MYB4 complex into the nucleus. Here, we show that the conserved GY/FDFLGL motif contributes to the interaction between MYB factors and SAD2. The Asp → Asn mutation in the GY/FDFLGL motif abolishes the interaction between MYB transcription factors and SAD2, and therefore they cannot be transported into the nucleus and cannot repress their target genes. We found that MYB4(D261N) loses the capacity to repress expression of the cinnamate 4-hydroxylase (C4H) gene and biosynthesis of sinapoyl malate. Our results indicate conservation among MYB transcription factors in terms of their interaction with SAD2. Therefore, the Asp → Asn mutation may be used to engineer transcription factors.

53 citations


"A Molecular Blueprint of Lignin Rep..." refers background in this paper

  • ...of AtMYB4, AtMYB7, and AtMYB32, thereby increasing their repression of their target genes [41,42]....

    [...]

Journal ArticleDOI
TL;DR: The results indicate that FtMYB11 acts as a regulator via interacting with FtSAD2 or FtJAZ1 to repress phenylpropanoid biosynthesis, and this repression depends on two conserved Asp residues of its SID-like motif.
Abstract: Summary Little is known about the molecular mechanism of the R2R3-MYB transcriptional repressors involved in plant phenylpropanoid metabolism. Here, we describe one R2R3-type MYB repressor, FtMYB11 from Fagopyrum tataricum. It contains the SID-like motif GGDFNFDL and it is regulated by both the importin protein ‘Sensitive to ABA and Drought 2’ (SAD2) and the jasmonates signalling cascade repressor JAZ protein. Yeast two hybrid and bimolecular fluorescence complementation assays demonstrated that FtMYB11 interacts with SAD2 and FtJAZ1. Protoplast transactivation assays demonstrated that FtMYB11 acts synergistically with FtSAD2 or FtJAZ1 and directly represses its target genes via the MYB-core element AATAGTT. Changing the Asp122 residue to Asn in the SID-like motif results in cytoplasmic localization of FtMYB11 because of loss of interaction with SAD2, while changing the Asp126 residue to Asn results in the loss of interaction with FtJAZ1. Overexpression of FtMYB11or FtMYB11D126N in F. tataricum hairy roots resulted in reduced accumulation of rutin, while overexpression of FtMYB11D122N in hairy roots did not lead to such a change. The results indicate that FtMYB11 acts as a regulator via interacting with FtSAD2 or FtJAZ1 to repress phenylpropanoid biosynthesis, and this repression depends on two conserved Asp residues of its SID-like motif.

53 citations


"A Molecular Blueprint of Lignin Rep..." refers background in this paper

  • ...MYB subgroup 4 EAR motif pdLNL(D/E)Lxi(G/S) AATAGTT [23]...

    [...]

  • ...The subgroup 4 FtMYB11 binds to the AATAGTT motif to repress the expression of FtPAL, FtC4H, and Ft4CL (Figure 2 and Table 1)....

    [...]

  • ...through the 26S proteasome pathway [23,43]....

    [...]

Journal ArticleDOI
TL;DR: The characterization of 81 pairs of Populus R2R3-MYB genes showed that these gene pairs resulted from multiple types of gene duplications and had five different gene fates.
Abstract: In plants, the R2R3-MYB gene family contains many pairs of paralogous genes, which play the diverse roles in developmental processes and environmental responses. The paper reports the characterization of 81 pairs of Populus R2R3-MYB genes. Chromosome placement, phylogenetic, and motif structure analyses showed that these gene pairs resulted from multiple types of gene duplications and had five different gene fates. Tissue expression patterns revealed that most duplicated genes were specifically expressed in the tissues examined. qRT-PCR confirmed that nine pairs were highly expressed in xylem, of which three pairs (PdMYB10/128, PdMYB90/167, and PdMYB92/125) were further functionally characterized. The six PdMYBs were localized to the nucleus and had transcriptional activities in yeast. The heterologous expression of PdMYB10 and 128 in Arabidopsis increased stem fibre cell-wall thickness and delayed flowering. In contrast, overexpression of PdMYB90, 167, 92, and 125 in Arabidopsis decreased stem fibre and vessel cell-wall thickness and promoted flowering. Cellulose, xylose, and lignin contents were changed in overexpression plants. The expression levels of several genes involved in secondary wall formation and flowering were affected by the overexpression of the six PdMYBs in Arabidopsis. This study addresses the diversity of gene duplications in Populus R2R3-MYBs and the roles of these six genes in secondary wall formation and flowering control.

52 citations


"A Molecular Blueprint of Lignin Rep..." refers background in this paper

  • ...Overexpression of the poplar xylem-specific TFs PdMYB90, PdMYB167 (orthologs of AtMYB52), PdMYB92, and PdMYB125 decreases the expression of xylan, lignin, and cellulose biosynthetic genes, modifying stem cell wall composition [57]....

    [...]

Journal ArticleDOI
TL;DR: It is shown that reversible monophosphorylation at Ser123 or Ser125 acts as an on/off switch for the activity of 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem-differentiating xylem of Populus trichocarpa.
Abstract: Although phosphorylation has long been known to be an important regulatory modification of proteins, no unequivocal evidence has been presented to show functional control by phosphorylation for the plant monolignol biosynthetic pathway. Here, we present the discovery of phosphorylation-mediated on/off regulation of enzyme activity for 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem-differentiating xylem (SDX) of Populus trichocarpa. Phosphorylation turned off the PtrAldOMT2 activity, as demonstrated in vitro by using purified phosphorylated and unphosphorylated recombinant PtrAldOMT2. Protein extracts of P. trichocarpa SDX, which contains endogenous kinases, also phosphorylated recombinant PtrAldOMT2 and turned off the recombinant protein activity. Similarly, ATP/Mn2+-activated phosphorylation of SDX protein extracts reduced the endogenous SDX PtrAldOMT2 activity by ∼60%, and dephosphorylation fully restored the activity. Global shotgun proteomic analysis of phosphopeptide-enriched P. trichocarpa SDX protein fractions identified PtrAldOMT2 monophosphorylation at Ser123 or Ser125 in vivo. Phosphorylation-site mutagenesis verified the PtrAldOMT2 phosphorylation at Ser123 or Ser125 and confirmed the functional importance of these phosphorylation sites for O-methyltransferase activity. The PtrAldOMT2 Ser123 phosphorylation site is conserved across 93% of AldOMTs from 46 diverse plant species, and 98% of the AldOMTs have either Ser123 or Ser125. PtrAldOMT2 is a homodimeric cytosolic enzyme expressed more abundantly in syringyl lignin-rich fiber cells than in guaiacyl lignin-rich vessel cells. The reversible phosphorylation of PtrAldOMT2 is likely to have an important role in regulating syringyl monolignol biosynthesis of P. trichocarpa.

51 citations