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Journal ArticleDOI

A Molecular Blueprint of Lignin Repression

01 Nov 2019-Trends in Plant Science (Trends Plant Sci)-Vol. 24, Iss: 11, pp 1052-1064

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.

AbstractAlthough lignin is essential to ensure the correct growth and development of land plants, it may be an obstacle to the production of lignocellulosics-based biofuels, and reduces the nutritional quality of crops used for human consumption or livestock feed. The need to tailor the lignocellulosic biomass for more efficient biofuel production or for improved plant digestibility has fostered considerable advances in our understanding of the lignin biosynthetic pathway and its regulation. Most of the described regulators are transcriptional activators of lignin biosynthesis, but considerably less attention has been devoted to the repressors of this pathway. We provide a comprehensive overview of the molecular factors that negatively impact on the lignification process at both the transcriptional and post-transcriptional levels.

Topics: Lignocellulosic biomass (52%)

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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Review
A Molecular Blueprint of Lignin Repression
Marc Behr,
1
Gea Guerriero,
2
Jacqueline Grima-Pettenati,
3
and Marie Baucher
1,
*
Although lignin is es sential to ensure the correct growth and development of land plants, it may
be an obstacle to the production of lignocellulosics-based biofuels, and reduces the nutritional
quality of crops used for human consumption or livestock feed. Th e need to tailor the lignocel-
lulosic biomass for more effi cient biofuel production or for improved plant digestibility has
fostered considerable advances in our understanding of th e lignin biosynthetic pathway and
its regulation. Most of the described regulators are transcriptiona l activators of lignin biosyn-
thesis, but considerably less attention ha s been devoted to the repr essors of this pathway .
We provide a comp rehensive overview of the molecular factors that negatively impact on the
lignification process at both the transcriptional and post-transcriptional levels.
Challenging Lignin
Lignin is a major cell wall component that f ulfill s fundamental fu nctio ns in plant develo pment as well
as in defense against pests and pathogens. This heteropolymer impregnates the compound middle
lamella and the secondary cell walls (SCWs) of cells genetically programmed to be lignified, such as
xylem tracheary elements as well as xylem and phloem fibers. Lignin biosynthesis spans the phenyl-
propanoid and the monoli gnol pathways, from phenylalanine to p-cou ma ryl, coniferyl, and sinap yl
alcohols. Monolignol homeostasis is regulated through a balance between the molecular factors
that an tago nisti cally regulate their biosyn thesis . After monoli gnols are exc reted into the apoplast,
they are oxidized by the phenol-oxidoreductase laccases and/or by class III perox idases, and are
polymerized by radical coupling. A complex regulatory network involving phytohormones, transcri p-
tion factors (TFs), and post-transcriptional events guide SCW deposition and lignification [1].This
network prevents lignin deposition in tissues undergoi ng active divisi on or elongation such as apical
meristems, vascular cambium, and immature x ylem cells, as well as in non-lignified tissues such as
stem pith and flax or hemp bast fibers that harbor gelatinous walls under normal devel opmental con-
ditions. Similarly, this network negatively regulates lignification in xylem tissue formed i n response to
mechanical stresses such as, for instance, poplar tension wood that harbors hypolignified gelatinous
walls.
The physical and chemical properties of lignin downgrade the value of plant feedstocks in several ap-
plications (Box 1). Ligni n is known to impair pulping [2] and to reduce forage digestibility by ruminants
[3,4]. It negatively impacts on the saccharification of plant biomass, therefore hampering biofuel pro-
duction [5], even though lignin content and composition did not always correlate with saccharification
potentia l [6,7]. I n add itio n, a high lignin content in crops used in the human d iet d ecreases their nutri-
tional quality [8]. Various environmental stresses are known to induce ligni n biosynthesis [9,10].Inthe
context of global climate change , a higher ligni n content in crops and fo rest trees may be expected,
thereby negatively affecting their c hemical composition for various end-uses. Hence, a better under-
standing of th e molecular mechanisms repressing ligni fication will be important to secure food qual-
ity and inspire biotechnological strategies for lignocellulosic biomass transformation. In light of these
crucial societal needs, this review summarizes the main transcriptional events leading to the repres-
sion of li gnification in eudicots. Examples of post-t ranscri ption al events negatively regulating mono-
lignol biosynthesis and lignin polymerization are also discussed. The regulation of lignification in
monocots and/or grasses dis plays several divergences; these were r ecent ly reviewed [1,11] and will
not be discussed in this article.
Transcriptional Repression of Lignin Biosynthesis
The roles of transcriptional activators promoting lignin biosynthesi s have been well documented in
various plants, such as thale c ress (Arabidopsis thaliana)(reviewedin[12]) and tree species (re vi ewed
1
Laboratoire de Biotechnologie Ve
´
ge
´
tale,
Universite
´
libre de Bruxelles, 6041
Gosselies, Belgium
2
Environmental Research and Innovation
Department, Luxembourg Institute of
Science and Technology, 4422 Belvaux,
Luxembourg
3
Laboratoire de Recherche en Sciences
Ve
´
ge
´
tales, Centre National de la
Recherche Scientifique (CNRS) Universite
´
Paul Sabatier Toulouse III (UPS), 31326
Castanet-Tolosan, France
*Correspondence: mbaucher@ulb.ac.be
Highlights
Negative regulators of lignin
biosynthesis are promising tools to
tailor biomass properties to meet
human requirements.
The Mediator complex adds
another level of transcription regu-
lation to the several transcription
factors that are known to repress
lignification.
Targeted transcript or protein
degradation, as well as protein–
protein interactions, fine-tune the
spatiotemporal pattern of
lignification.
Understanding the interactions
between genes, non-coding RNAs,
and proteins opens new avenues
towards understanding secondary
cell wall formation.
1052 Trends in Plant Science, November, Vol. 24, No. 11 https://doi.org/10.1016/j.tplants.2019.07.006
ª 2019 Elsevier Ltd. All rights reserved.
Trends in Plant Science

in [13,14]). The negative regulat ors of li gni n biosynt hesis an d thei r und erly ing dir ectin g networks are
tightly controlled.
NAC TFs, the Two Sides of SCW Regulation
Members of the NAC family act as first- a nd second-level master swi tches in the regulati on of a bat-
tery of downstream TF s and SCW bio synt heti c genes [15–18]. Among these, VASCU LAR-RE LATE D
NAC DOMAINs(VNDs) and NAC SECONDARY WALL THICKENING PROMOTING FACTORs
(NSTs), also called SECONDARY WALL-ASSOCIATED NAC DOMAINs(SNDs), are positive regula-
tors of SCW thickening (reviewed in [15]). In thale cress, VNDs induce differentiatio n (see Glossary)
of vessels, whereas NSTs/SNDs regulate SCW dep osition in fibers. The activities of these regulators
are tightly controlled by different mechanisms [16–21].
XYLEM NAC DOMAIN 1 (XND1) blocks tracheary element differentiation through the negative regu-
lation of VND7 [19,20], preventing prec ocious deposition of the SCW (Figur e 1, Key Figure). This
repression is possibly linked to cell differentiation because XND1 physically interacts with the cell cy-
cle and differentiation regulator RETINOBLASTOMA-RELATED via its LXCXE and LXCXE-mimic mo-
tifs [20] .
VND-INTERACTING 2 (VNI2) is a transcriptional repressor reported to regulate the timing and spatial
regulation of xylem cell de velopmen t [21]. VNI2 acts as a passive repressor by forming he tero dimer s
with VND proteins, preventing their positive regulation of vessel differentiation (notably through the
expression of lignin-related AtMYB46). Because VNI2 displays an ethylene-responsive element bind-
ing f actor- associate d amphiphilic repression (EAR)- like motif [pdLNL(D/E) Lxi(G /S)], it might also
actively repress the ge nes involved in xylem diffe rentiat ion [21].
The SCW activator NST2 is negatively transcriptionally regulated by WRKY12 (Figure 1), which binds
to the W-box cis -ele ment in the NST2 promoter region (Table 1) [25]. WRKY12 and its functional pop-
lar (Populus trichocarpa)orthologPtrWRKY19 wereshowntobenegativeregulatorsofSCWdeposi-
tion in the pith [25,28].
In poplar, the transcriptional activities of SNDs and VNDs are negatively regulated by dedicated
splice variants with retained intron sequences [29]. An intron-retained (IR) splice variant PtrVND6-
C1
IR
negatively regulates the expression of PtrMYB021 (a poplar ortholog of AtMYB46)byforming
heterodimers with the full-size PtrVND6s, suppressing their positive transcriptional activity (Fi gure 1).
In addition, PtrVND 6-C 1
IR
downregulates the expression of five f ull-size PtrVND6s. Similarly,
PtrSND1-A2
IR
dimerizes with full-size PtrSND1s. Importantly, PtrVND6-C1
IR
and PtrSND 1-A 2
IR
cannot
suppress their cognate TFs but can su ppress al l members of the other family, indicating that the splice
variants from the PtrVND6 and PtrSND1 family may exert reciprocal crossregulation for complete
transcriptional regulation of these two families in wood formation, providing a higher level of regu-
lation to maintain homeostasis in plants to avoid abnormal growth and development [29].
Box 1. Handling Lignification
Lignin lowers the digestib ili ty of several feed, food, and biomass p roducts, resulting in detrimental effects that
may worsen in the context of global warming.
Attempts to reduce lignin content in model plants through targeted gene expression modification have often
resulted in impaired growth with a subsequent yield penalty.
Although several molecular factors ca n enhance lignin b iosynthesis, those preventing/inhibiting this develop-
mental process are currently under investigation.
Negative regulation of lignin biosynthesis is achieved through diverse mechanisms ranging from DNA acces-
sibility to targeted proteolysis.
Glossary
Cis-element:acis-element is a
conserved nucleotide sequence
(e.g., the G-box) that is generally
found in the promoter region of
the regulated gene, and is recog-
nized by a specific family or sub-
family of transcription factors (TFs;
e.g., MYBs). These TFs are trans-
regulating elements because they
tune the expression of specific
genes through intermolecular in-
teractions. Mediator proteins are
also trans-regulating elements.
Differentiation:asuiteofincre-
mental cellular and molecular
modifications leadi ng to the for-
mation of specialized groups of
cells from undifferentiated tissues,
such as the shoot apical meristem
or the vascular cambium. The
process, the timing, and the loca-
tion of differentiation are under
stringent genetic regulation.
Lignification is a key feature of the
differentiation of vessels and fi-
bers in xylem a nd phloem.
Heterodimer: in this review this
term designates a complex of two
different proteins, usually TFs.
Heterodimer formation changes
the properties of a p rotein relative
to those of the isolated protein.
Heterodimerization may for
instance lead to higher transcrip-
tional activity (either activation o r
repression), result in decreased
activity of one of the proteins in
the complex, or target one of the
proteins to a specific cellular
compartment (e.g., the nucleus or
cytosol).
Non-coding RNA (ncRNA):an
RNA that is not translated into
protein. The functions of ncR NAs
range from transcript degradation
(microRNAs) to translation regu-
lation, gene expression or RNA
splicing. We here review some
long non-coding RNAs (lncRNAs)
and microRNAs mediating the
degradation of laccase transcripts.
Semidominant mutant:analleleis
said to be semidominant when the
heterozygote displays an pheno-
type intermediate between that of
the homozygote mutant (null
mutant) and the wild type. Such
mutants are useful to investigate
the function of a gene whose null
mutation results in a severe
phenotype such as dwarfism or
lethality. Semidominance is syn-
onymous with ’Incomplete
dominance’.
Trends in Plant Science, November, Vol. 24, No. 11 1053
Trends in Plant Science

Key Figure
Molecular Regulation of Lignin Repression in Dicots at the
Chromatin, Transcriptional, Post-Transcriptional, and Post-
Translational levels (green boxes)
Chromatin–mediated repression
EgMYB1
Transcriptional repression
AtMED5
AtPAL1/2
AtC4H
At4CL1
AtMYB4
AtKFB01/39/50
MYB
WRKY
NAC
Subgroup 4
AtMYB3
AmMYB308
AmMYB330
AtMYB4/EgMYB1/PdMYB221
AtMYB7
AtMYB32
AtγMYB2
AtMYB52
AtMYB75
PttMYB21a
PdMYB90, PbMYB167
PdMYB92, PdMYB125
BELL family
AtBLH6
KNOX family
AtBP
PtaARK2
AtKNAT7
Interactions KNAT7
KNAT7–MYB75
KNAT7–DRIF1
KNAT7–OFP
KNAT7–BLH6
KNAT7–BLH6–OFP
AtWRKY12 / PtrWRKY19
AtNST2
Post–transcriptional
repression
miR397
Post–translational regulation
PAL
KFB01
KFB20
KFB39
KFB50
U
U
PAL
26S proteasome
Reduced DNA accessibility through chromatin condensation
HD–ZIP
TALE
AtHB15/PtrPCN
/PtrHB11
miR857
PtrVND6–C1
IR
, PtrSND1–A2
IR
PtrVND6, PtrSND1
AP2
EjAP2-1–EjbHLH1–EjMYBs
PsnSHINE2
PtrhAT
PtrMYB021
Transcriptional complex
LAC
AtVNDs AtVND7
AtVNI2 AtXND1
Active
PtrAldOMT2
P
Ser
123
Ser
125
Inactive
PtrAldOMT2
LTF1
Phosphorylated
LTF1
U
EgH1.3
Tre nd s
T rends
in
in
Plant
Plant
Science
Science
Splice variants: these arise from
alternative RNA splicing events,
and display an mRNA sequence
that differs from that of the regular
transcript, resulting in a different
protein. Alternative splicing allows
the biosynthesis of several pro-
teins from a single coding gene. In
this review we describe alterna-
tively spliced proteins regula ting
the expression of clos ely rela ted
coding genes.
(Key figure legend at the bottom of the next page.)
1054 Trends in Plant Science, November, Vol. 24, No. 11
Trends in Plant Science

R2R3 MYBs, the Gateke eper s of SCW Formation and Lignificati on
Some members of the R2R3-MYB TF family positively regulate gene expression of phenylpropanoid
and ligni n biosyntheti c 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 highl ighted in a
recent review [33].
The first MYB factors shown to repress lignin biosynthesis were AmMYB308 and AmMYB330 from An-
tirrhinum majus [34] . Overexpression of th ese genes in to bacco caused a decrease in xylem l ignin
content by reducing the expression of phenylpropanoid and lignin biosynt hetic genes (C4H, 4CL,
and CAD). The knockout of AtMYB4, a thale cress ortholog of AmMYB308, displayed increased
amounts of sinapate esters t hrough increased expression of C4H [35]. AtMYB4 belongs to subgroup
4 and, a s the other proteins from this subgro up (AtMYB3, AtMYB7, and AtMYB32), contains an EAR-
like repression motif in its C-terminus [36]. Members of subgroup 4 repress the phenylpropanoid
pathway, the lign in pathway, a nd/or the bi osynth esis of pigmen ts (Figure 1) [35,37]. AtMYB 4 is down-
regulated in thale cress ectopic lignification de-etiolated 3, pom-pom 1,andectopic lignification 1
mutants [38], suggesting that it could negatively regulate lignin biosynthesis. AtMYB32 negatively
controls the expression o f genes involv ed in pheny lpropano id and lignin bio synthe sis, affecting
the pollen-wall composition [39]. The repressive activity of subgroup 4 is tightly con troll ed. AtMYB7,
AtMYB32, and AtMYB4 negatively regulate their own transcription as well as that of AtMYB52 [40].
SENSITIVE TO ABA AND DROUGHT 2 (SAD2), an impor tin b-like protein, mediates nuclear trafficking
of AtMYB4, AtMY B7, a nd AtMYB 32, ther eby i ncre asing th eir repr essi on of t heir t arget genes [41,42].
The in teracti on between SAD2 and members of subgroup 4 occurs through thei r SID domain
GXXDFxxxG/DL, w hich is also a signatu re for protein degradation thr ough the 26S proteasome
pathway [43]. In buckwheat (Fagopyrum tataricum), jasmonates induce not only the expression of
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, s uch 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 histonevariant;hAT,hoboactivatorTam3transposase;HB,
homeobox; KFB Kelch F-box; KNAT, knotted1-like TALE homeodomain; LAC, laccase; LTF, l ignin biosynthesis-
associated transcription facto r; MED, Mediator; NST, NAC seconda ry wall thickening promoting f actor; 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/).
Protein Binding domain Binding site Refs
TALE WFIN residue of homeodomain KN-1 TGACAG(G/C)T [22]
MYB subgroup 4 EAR motif pdLNL(D/E)Lxi(G/S) AATAGTT [23]
AP2/ERF Acidic C-terminal domain SNBE, SMRE, GCC-box [24]
WRKY WRKYGQK residue W-box TTGACT/C [25]
miRNA
LAC
Not applicable Cu-oxidase domain [26]
KFB
PAL
Kelch domain at the C-terminus Unknown [27]
Table 1. Binding Sites of Proteins Involved in t he Repression of Monolignol Biosynthesis
Trends in Plant Science, November, Vol. 24, No. 11 1055
Trends in Plant Science

SAD2 and of five members of subgroup 4, but also the degradation of the corresponding proteins
through the 26S prot easome pathway [23,43]. The subgroup 4 FtMYB11 binds to the AATAGTT moti f
to repress the expression of FtPAL, FtC4H,andFt4CL (Figure 2 and Table 1).Therepressionmech-
anism of AtMYB3 is completely different from that of AtMYB4, AtMYB7, and AtMYB32 because it is
directly regulated by the corepressors NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1
(LNK1) and LNK2, which could facilitate binding of A tMYB3 to the C4H promoter [37] .
More direct evidence of the negative regulation of l ignin biosynthesis by potential orthologs of At -
MYB4 has been reported in species other than thale cress (Figure 1). Overexpression of Eucaly ptus
gunni EgMYB1 in thale cress and poplar negatively regulates SCW formation, including lignin biosyn-
thesis [45]. When interacting with the linker histone variant EgH1.3, EgMYB1 displays stronger repres-
sive activity, thereby preve nting premature lignification of xylem cel ls during their early st ages of dif-
ferenti ati on [46]. The two orthologs of EgMYB1 in Popu lus del toid es and P. tomentosa (PdMYB221
and PtoMYB156) also reduce the SCW thickness o f xylem fibers and the content of cellul ose, lignin,
and xylan [47,48]. Transgenic poplars overexpressing both PtrMYB221 and GA20-oxidase,anenzyme
involved in gibberellin biosynthesis, showed a twofold increase in plant biomass, a 16% reduction of
lignin conte nt, a h igher amou nt o f holo cellu lose, and an 8% improved s acchari ficatio n effici ency
compared to the wild type [49]. In addit ion, PtoMYB156 represses phenylpropanoid biosynthetic
genes, leading to a reduction in total phenolics and flavonoids [48].
During the formation of cotton ovule/fiber SCW, orthologs of AtMYB4 are also highly expressed, sup-
porting their r oles in repressing lignification [50]. In hemp, a motif re cognized by MYB3 was found in
the promoter sequence of the FA SCICLI N-LI KE ARA BINOGALA CTAN (FLA) genes that are highly e x-
pressed in mature phloem fibers. Those FLAs may be under a MYB3-driven reg ul ato ry circuit deter-
mining bast fiber hypolignification [51].
In loquat (Eriobotrya japonica), EjMYB2, a member of subgroup 4, represses the e xpressio n of gen es
belonging t o the phenylpropanoid and lignin pathways (su ch a s HCT, CC oAOMT ,andCCR1),
whereas EjMYB1 mediat es their up regul ation [52] .EjMYB2interactswithEjbHLH1andwithamember
of the AP2/ERF TF family, EjAP2-1, to repress cold-induced lignification (Figure 1) [53]. EjMYB1 and
EjMYB2 both interact with EjAP2-1 to regulate the expression of Ej4CL1, le ading t o a str onger repres-
sive activity of EjMYB2 and attenuation of the EjMYB1-mediated activation [54]. Although EjAP2-1 has
two EAR motifs, it is unable to bind directly to the promoters of the lignin biosynthetic genes, sug-
gesting that its repressive activity occurs via protein–protein interactions with MYB proteins [54].
On the other hand, overe xpressi on of anothe r member of the AP2/ERF family, Psn SHINE2 from
P. simonii 3 P. nigra, in tobacco represses the expression of MYB and NAC activators of the lignin
pathway, leading to reduced l igni n content in stems [24] . PsnSHINE2 bi nds not only to the A P2/
ERF binding box but also to a secon dary wall NAC binding element (SNBE, motif
TCCTTTTCTCTCTA AGCAT ) and to three MYB-binding elements (SMBEs; motifs ACCAAAT,
ACCTACC, and ACCAACC) [24] that are present in the promoters of lignin, xylan, and cellulose
biosynthetic genes (Table 1) [55]. A P2/ERF are therefore able to regul ate t he lignin biosynthet ic
pathway both directly and indirectly.
Other MYBs not belonging to subgroup 4 negatively regulate the expression of genes of the mono-
lignol pat hway (Figure 1). Several Atmyb52 insertion lines show strong accumulation of lignin in inter-
fascicular fibers, metaxylem vessels, and phloem cap cells [56]. MYB52 is coexpressed with several TFs
regulating SCW formation (MYB85, SND2, XND1) and genes involved in cellulose and xylan biosyn-
thesis [56], highl ighting the import ance of monolign ol homeostasis in lign ifying tissu es. Overexpres-
sion of the poplar xylem-specific TFs PdMYB90, PdMYB167 (orthologs of AtMYB52), PdMYB92,and
PdMYB125 decreases t he e xpressio n of xylan, lignin, and cellulose bio synthe tic genes, modifying
stem cell wall composition [57]. Downregulation of PttMYB21a (homolog to AtMYB52)byusingan
antisense strategy in transgenic aspen (Populus tremula 3 P. tremuloides) increased lignin content
while decreasing the pool of structural carbohydrates in the bark [58]. However, anoth er PttMYB21a
antisense line showed a lower lignin content in wood, a decreased stem height and diameter, as well
as increased glucose release during enzymatic digestion relative to the wild type [7], highlighting the
1056 Trends in Plant Science, November, Vol. 24, No. 11
Trends in Plant Science

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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.

10 citations


Journal ArticleDOI
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.
Abstract: Breeding to enrich lignin, a major component of lignocelluloses, in plants contributes to enhanced applications of lignocellulosic biomass into solid biofuels and valuable aromatic chemicals. To collect information on enhancing lignin deposition in grass species, important lignocellulose feedstocks, we generated rice (Oryza sativa) transgenic lines deficient in OsWRKY36 and OsWRKY102, which encode putative transcriptional repressors for secondary cell wall formation. We used CRISPR/Cas9-mediated targeted mutagenesis and closely characterized their altered cell walls using chemical and nuclear magnetic resonance (NMR) methods. Both OsWRKY36 and OsWRKY102 mutations significantly increased lignin content by up to 28 % and 32 %, respectively. Additionally, OsWRKY36/OsWRKY102-double-mutant lines displayed lignin enrichment of cell walls (by up to 41 %) with substantially altered culm morphology over the single-mutant lines as well as the wild-type controls. Our chemical and NMR analyses showed that 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 lignins. Our results provide evidence that both OsWRKY36 and OsWRKY102 are associated with repression of rice lignification.

4 citations


Journal ArticleDOI
TL;DR: Findings implicate important roles for MYB transcription factors in coordinated regulation of grass lignin biosynthesis including γ-acylated and tricin-incorporated lign in biosynthesis.
Abstract: Recent analyses of cell wall components of various grass mutant and transgenic lines have provided information on characteristic transcriptional regulation of cell wall formation in grasses, although its knowledge yet remains limited compared with that for eudicot cell wall formation. MYB transcription factors, which are regarded as downstream regulators operating under NAM, ATAF1/2, and CUC2 (NAC) domain transcription factors, have been suggested to be involved in direct regulation of cell wall biosynthesis. In this review, we discuss MYB-mediated transcriptional regulation of the biosynthesis of grass lignins, including grass-specific lignin components such as γ-acyl groups and flavone tricin units. Grass mutant and transgenic lines harboring modified cell-wall-associated MYB genes display altered composition of the γ-acylated and tricin-incorporated lignin units and/or modified expression of enzyme genes involved in the formation of these grass-specific lignin components along with conserved monolignol biosynthetic genes. These findings implicate important roles for MYB transcription factors in coordinated regulation of grass lignin biosynthesis including γ-acylated and tricin-incorporated lignin biosynthesis.

3 citations


Journal ArticleDOI
Zexiong Chen1, Ning Tang1, Li Huihe1, Guohua Liu1, Ling Tang 
TL;DR: Investigating the changes of the cell wall components, hormones and transcriptome profiles during rhizome development provides new insight into molecular events of cellulose accumulation mainly mediated by hormones and transcription factors in ginger rhizomes.
Abstract: Ginger is a popular vegetable crop primarily consumed in Chongqing and Sichuan, China. The rhizome becomes tough and fibrous rapidly with the tissue maturation, resulting in loss of edible quality. To characterize biochemical and molecular mechanisms of rhizome texture modification, we investigated the changes of the cell wall components, hormones and transcriptome profiles during rhizome development. With rhizomes maturation, the contents of cellulose and hemicellulose increased gradually, and no obvious change was observed in lignin accumulation. The levels of ABA and cytokinin exhibited a gradual decline during maturation of ginger rhizome, which were negatively correlated with cellulose content, while GA3 displayed an increasing trend and a positive correlation with cellulose production. Transcriptomic analysis identified candidate genes involved in cellulose biosynthesis including cellulose synthase subunit A (CesA) genes, hormone metabolism and signaling genes, and NAC-MYB-based transcription factors. Further analysis demonstrated ZoCESA7, ZoNAC16 and ZoMYB37/39/41, homologs of AtCESA7, AtVND6/7 and AtMYB46/83, respectively, positively correlated with cellulose contents, whereas ZoNAC3/9/13 and ZoMYB7/11/18, homologs of AtVND6/7 and AtMYB4, respectively, negatively correlated with cellulose levels, indicating their involvements in cellulose synthesis during rhizome development. These findings provide new insight into molecular events of cellulose accumulation mainly mediated by hormones and transcription factors in ginger rhizomes.

1 citations


Journal ArticleDOI
Abstract: BACKGROUND Phosphorylation modification, one of the most common post-translational modifications of proteins, widely participates in the regulation of plant growth and development. Fibers extracted from the stem bark of ramie are important natural textile fibers; however, the role of phosphorylation modification in the growth of ramie fibers is largely unknown. RESULTS Here, we report a phosphoproteome analysis for the barks from the top and middle section of ramie stems, in which the fiber grows at different stages. A total of 10,320 phosphorylation sites from 9,170 unique phosphopeptides that were assigned to 3,506 proteins was identified, and 458 differentially phosphorylated sites from 323 proteins were detected in the fiber developmental barks. Twelve differentially phosphorylated proteins were the homologs of Arabidopsis fiber growth-related proteins. We further focused on the function of the differentially phosphorylated KNOX protein whole_GLEAN_10029667, and found that this protein dramatically repressed the fiber formation in Arabidopsis. Additionally, using a yeast two-hybridization assay, we identified a kinase and a phosphatase that interact with whole_GLEAN_10029667, indicating that they potentially target this KNOX protein to regulate its phosphorylation level. CONCLUSION The finding of this study provided insights into the involvement of phosphorylation modification in ramie fiber growth, and our functional characterization of whole_GLEAN_10029667 provide the first evidence to indicate the involvement of phosphorylation modification in the regulation of KNOX protein function in plants.

1 citations


References
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Journal ArticleDOI
TL;DR: An Arabidopsis thaliana line that is mutant for the R2R3 MYB gene, AtMYB4, shows enhanced levels of sinapate esters in its leaves, indicating that derepression is an important mechanism for acclimation to UV‐B in A.thaliana.
Abstract: An Arabidopsis thaliana line that is mutant for the R2R3 MYB gene, AtMYB4, shows enhanced levels of sinapate esters in its leaves. The mutant line is more tolerant of UV-B irradiation than wild type. The increase in sinapate ester accumulation in the mutant is associated with an enhanced expression of the gene encoding cinnamate 4-hydroxylase, which appears to be the principal target of AtMYB4 and an effective rate limiting step in the synthesis of sinapate ester sunscreens. AtMYB4 expression is downregulated by exposure to UV-B light, indicating that derepression is an important mechanism for acclimation to UV-B in A.thaliana. The response of target genes to AtMYB4 repression is dose dependent, a feature that operates under physiological conditions to reinforce the silencing effect of AtMYB4 at high activity. AtMYB4 works as a repressor of target gene expression and includes a repression domain. It belongs to a novel group of plant R2R3 MYB proteins involved in transcriptional silencing. The balance between MYB activators and repressors on common target promoters may provide extra flexibility in transcriptional control.

722 citations


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

  • ...The knockout of AtMYB4, a thale cress ortholog of AmMYB308, displayed increased amounts of sinapate esters through increased expression of C4H [35]....

    [...]

  • ...The repression mechanism of AtMYB3 is completely different from that of AtMYB4, AtMYB7, and AtMYB32 because it is directly regulated by the corepressors NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1 (LNK1) and LNK2, which could facilitate binding of AtMYB3 to the C4H promoter [37]....

    [...]

  • ...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]....

    [...]

  • ...Molecular Regulation of Lignin Repression in Dicots at the Chromatin, Transcriptional, Post-Transcriptional, and PostTranslational levels (green boxes) Chromatin–mediated repression EgMYB1 Transcriptional repression AtMED5 AtPAL1/2 AtC4H At4CL1 AtMYB4 AtKFB01/39/50 MYB WRKY NAC Subgroup 4 AtMYB3 AmMYB308 AmMYB330 AtMYB4/EgMYB1/PdMYB221 AtMYB7 AtMYB32 AtγMYB2 AtMYB52 AtMYB75 PttMYB21a PdMYB90, PbMYB167 PdMYB92, PdMYB125 BELL family AtBLH6 KNOX family AtBP PtaARK2 AtKNAT7 Interactions KNAT7 KNAT7–MYB75 KNAT7–DRIF1 KNAT7–OFP KNAT7–BLH6 KNAT7–BLH6–OFP AtWRKY12 / PtrWRKY19 AtNST2 Post–transcriptional repression miR397 Post–translational regulation PAL KFB01 KFB20 KFB39 KFB50 U U PAL 26S proteasome Reduced DNA accessibility through chromatin condensation HD–ZIP TALE AtHB15/PtrPCN/PtrHB11 miR857 PtrVND6–C1IR, PtrSND1–A2IR PtrVND6, PtrSND1 AP2 EjAP2-1–EjbHLH1–EjMYBsPsnSHINE2 PtrhAT PtrMYB021 Transcriptional complex LAC AtVNDs AtVND7 AtVNI2 AtXND1 Active PtrAldOMT2 P Ser 123 Ser125 Inactive PtrAldOMT2 LTF1 Phosphorylated LTF1 U EgH1....

    [...]

  • ...Members of subgroup 4 repress the phenylpropanoid pathway, the lignin pathway, and/or the biosynthesis of pigments (Figure 1) [35,37]....

    [...]


Journal ArticleDOI
Abstract: Lignin is a polymer of phenylpropanoid compounds formed through a complex biosynthesis route, represented by a metabolic grid for which most of the genes involved have been sequenced in several plants, mainly in the model-plants Arabidopsis thaliana and Populus. Plants are exposed to different stresses, which may change lignin content and composition. In many cases, particularly for plant-microbe interactions, this has been suggested as defence responses of plants to the stress. Thus, understanding how a stressor modulates expression of the genes related with lignin biosynthesis may allow us to develop study-models to increase our knowledge on the metabolic control of lignin deposition in the cell wall. This review focuses on recent literature reporting on the main types of abiotic and biotic stresses that alter the biosynthesis of lignin in plants.

619 citations


Journal ArticleDOI
TL;DR: It is proposed that KNOX proteins may act as general orchestrators of growth-regulator homeostasis at the shoot apex of Arabidopsis by simultaneously activating CK and repressing GA biosynthesis, thus promoting meristem activity.
Abstract: The shoot apical meristem (SAM) is a pluripotent group of cells that gives rise to the aerial parts of higher plants. Class-I KNOTTED1-like homeobox (KNOX) transcription factors promote meristem function partly through repression of biosynthesis of the growth regulator gibberellin (GA). However, regulation of GA activity cannot fully account for KNOX action. Here, we show that KNOX function is also mediated by cytokinin (CK), a growth regulator that promotes cell division and meristem function. We demonstrate that KNOX activity is sufficient to rapidly activate both CK biosynthetic gene expression and a SAM-localized CK-response regulator. We also show that CK signaling is necessary for SAM function in a weak hypomorphic allele of the KNOX gene SHOOTMERISTEMLESS (STM). Additionally, we provide evidence that a combination of constitutive GA signaling and reduced CK levels is detrimental to SAM function. Our results indicate that CK activity is both necessary and sufficient for stimulating GA catabolic gene expression, thus reinforcing the low-GA regime established by KNOX proteins in the SAM. We propose that KNOX proteins may act as general orchestrators of growth-regulator homeostasis at the shoot apex of Arabidopsis by simultaneously activating CK and repressing GA biosynthesis, thus promoting meristem activity.

571 citations


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

  • ...with a lower bioactive pool of gibberellins [67]....

    [...]


Journal ArticleDOI
TL;DR: It is demonstrated that overexpression of two MYB genes from Antirrhinum represses phenolic acid metabolism and lignin biosynthesis in transgenic tobacco plants.
Abstract: MYB-related transcription factors are known to regulate different branches of flavonoid metabolism in plants and are believed to play wider roles in the regulation of phenylpropanoid metabolism in general. Here, we demonstrate that overexpression of two MYB genes from Antirrhinum represses phenolic acid metabolism and lignin biosynthesis in transgenic tobacco plants. The inhibition of this branch of phenylpropanoid metabolism appears to be specific to AmMYB308 and AmMYB330, suggesting that they recognize their normal target genes in these transgenic plants. Experiments with yeast indicate that AmMYB308 can act as a very weak transcriptional activator so that overexpression may competitively inhibit the activity of stronger activators recognizing the same target motifs. The effects of the transcription factors on inhibition of phenolic acid metabolism resulted in complex modifications of the growth and development of the transgenic plants. The inhibition of monolignol production resulted in plants with at least 17% less lignin in their vascular tissue. This reduction is of importance when designing strategies for the genetic modification of woody crops.

457 citations


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

  • ...The first MYB factors shown to repress lignin biosynthesis were AmMYB308 and AmMYB330 from Antirrhinum majus [34]....

    [...]


Journal ArticleDOI
TL;DR: The cloning of small RNAs from abiotic stressed tissues of Populus trichocarpa (Ptc) and the identification of 68 putative miRNA sequences that can be classified into 27 families based on sequence homology are reported, which suggests that the members of a family may have different functions.
Abstract: MicroRNAs (miRNAs), a group of small non-coding RNAs, have recently become the subject of intense study. They are a class of post-transcriptional negative regulators playing vital roles in plant development and growth. However, little is known about their regulatory roles in the responses of trees to the stressful environments incurred over their long-term growth. Here, we report the cloning of small RNAs from abiotic stressed tissues of Populus trichocarpa (Ptc) and the identification of 68 putative miRNA sequences that can be classified into 27 families based on sequence homology. Among them, nine families are novel, increasing the number of the known Ptc-miRNA families from 33 to 42. A total of 346 targets was predicted for the cloned Ptc-miRNAs using penalty scores of

445 citations


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

  • ...Their expression is developmentally regulated and/or under the control of external stimuli such as abiotic stress or nutrient availability [93,94]....

    [...]