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Wood, which represents the most abundant lignocellulosic biomass on earth, fulfils key roles in trees, and is also a raw material for multiple end-uses by mankind. The differentiation of this complex vascular tissue starts with cell division in the vascular cambium and is characterized by a massive deposition of lignified secondary cell walls mainly in fibres and vessels. A transcriptional network underlying this differentiation process ensures a tight regulation of the expression of thousands of genes both at the spatial and temporal levels. Most of our current knowledge of this hierarchical network was extrapolated from studies performed in Arabidopsis. Here, we review recent findings on the regulation of wood formation in angiosperm trees species highlighting conserved and distinct mechanisms with Arabidopsis. We provide examples shedding light on the central role of auxin and its cross-talk with other hormones at different stages of secondary xylem differentiation. Functional studies of trees’ wood-associated transcription factors revealed diversified functions as compared to their Arabidopsis orthologs. Sophisticated mechanisms of alternative splicing and cross-regulation between the two distinct groups of top level NAC-domain master regulators were uncovered. These findings underlie the high level of complexity of wood formation in trees and provide a framework for future lines of research in this exiting research field.
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Digging in wood: New insights
in the regulation of wood
formation in tree species
Eduardo L.O. Camargo
, Raphaël Ployet
, Hua Cassan-Wang
Fabien Mounet
, Jacqueline Grima-Pettenati
Laboratoire de Recherche en Sciences Vegetales, Universite de Toulouse, CNRS, UPS,
Castanet-Tolosan, France
´rio de Gen^omica e Expressa
˜o, Departamento de Genetica, Evoluc¸a
˜o e Bioagentes,
Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Brazil
*Corresponding author: e-mail address:
This paper is dedicated to the memory of Prof Carl Douglas, pioneer and mentor
in this research field who died prematurely.
1. Introduction 202
2. Hormonal control of wood formation 204
2.1 Reading the auxin gradient 204
2.2 The interplay between auxin and the WOX-CLE-PXY complex 205
2.3 The cross-talk between auxin and cytokinins stimulates cambium activity 206
2.4 The cross-talk between auxin and gibberellins promotes expansion
of cambial derivatives 207
2.5 Interaction between auxin and other hormones 210
2.6 Identifying new Aux/IAAs and ARFs as mediators of auxin signalling
in wood formation 211
2.7 Role of ethylene in xylem cell expansion and maturation 212
3. Main actors of the SW regulatory network: NACs and MYBs 212
3.1 Wood-associated NAC domain transcription factors 213
3.2 SW-associated MYB transcription factors 217
3.3 New NAC and MYB candidates putatively involved in the control of wood
formation in trees 220
4. Post-transcriptional and post-translational regulations of the SW regulators 223
4.1 Alternative splicing regulates cell wall thickening 223
4.2 Proteinprotein interactions 224
5. Concluding remarks 225
Acknowledgements 227
References 227
Further reading 233
Advances in Botanical Research, Volume 89 #2019 Elsevier Ltd
ISSN 0065-2296 All rights reserved.
Wood, which represents the most abundant lignocellulosic biomass on earth, fulfils key
roles in trees, and is also a raw material for multiple end-uses by mankind. The differ-
entiation of this complex vascular tissue starts with cell division in the vascular cambium
and is characterized by a massive deposition of lignified secondary cell walls mainly in
fibres and vessels. A transcriptional network underlying this differentiation process
ensures a tight regulation of the expression of thousands of genes both at the spatial
and temporal levels. Most of our current knowledge of this hierarchical network was
extrapolated from studies performed in Arabidopsis. Here, we review recent findings
on the regulation of wood formation in angiosperm trees species highlighting con-
served and distinct mechanisms with Arabidopsis. We provide examples shedding light
on the central role of auxin and its cross-talk with other hormones at different stages of
secondary xylem differentiation. Functional studies of treeswood-associated transcrip-
tion factors revealed diversified functions as compared to their Arabidopsis orthologs.
Sophisticated mechanisms of alternative splicing and cross-regulation between the
two distinct groups of top level NAC-domain master regulators were uncovered. These
findings underlie the high level of complexity of wood formation in trees and provide a
framework for future lines of research in this exiting research field.
1. Introduction
Wood, also called secondary xylem, is a highly specialized vascular
tissue characterized by the presence of thick heavily lignified secondary cell
walls composed of three main polymers: cellulose, hemicelluloses (e.g.
xylan) and lignin. Wood is the most abundant plant biomass on earth and
an immense reservoir of fixed carbon for long periods of time. It is used
for myriad applications such as construction, pulp and paper making and as
a source of renewable energy alternative to fossil fuels. Distinct wood prop-
erties are required for the different end-uses of wood as raw material and they
are not necessarily the properties which are beneficial for trees fitness and
adaptation to environment. Anatomical, chemical and physical properties
of wood are determined throughout a complex dynamic process called
xylogenesis controlled by both internal signals like hormones and external
signals occurring during the tree’s life (reviewed in Mauriatetal.,2014).
Wood is produced by the activity of a lateral meristem, the vascular cam-
bium that is responsible for both secondary growth and the perennial life of
trees (Dejardin et al., 2010). This particular meristem is composed of two
types of stem cells (the fusiform and the ray initials) that divide asymmetri-
cally to maintain the stem cell population and to produce daughter cells.
202 Eduardo L.O. Camargo et al.
Cambial cells division rate determines the rate of wood formation. In woody
angiosperms, the elongated fusiform initials differentiate into axially ori-
ented woody cells (fibres, vessels, axial parenchyma) ensuring water conduc-
tion and mechanical support for the plant body. The nearly isodiametric ray
initials give rise to transversely oriented ray parenchyma (contact and isola-
tion ray cells) ensuring transverse conduction and nutrient storage (reviewed
in Mellerowicz, Baucher, Sundberg, & Boerjan, 2001;Morris et al., 2016).
The differentiation of daughter cells into distinct mature xylem cell-types
with specialized functions involves sequential steps including cell expansion,
substantial deposition of thick lignified secondary cell walls (SWs),
programmed cell death and heartwood maturation (Zhong & Ye, 2015). This
temporally and spatially tightly controlled process has driven considerable
research attention because of the economic importance of wood.
Most of our current knowledge on the regulation of wood formation was
extrapolated from Arabidopsis thaliana (Schuetz, Smith, & Ellis, 2013), whose
secondary growth is limited to hypocotyl and roots. Despite the fact that this
annual plant undergoes true secondary growth (i.e. originating from cambial
activity) its secondary xylem lack the parenchyma-like files of cells, the so
called “rays”, one key feature of angiosperm wood (Chaffey, Cholewa,
Regan, & Sundberg, 2002). In contrast to herbaceous annual plants, trees
need to synthesize heavily thickened secondary cell walls, in a large and
stable quantity to withstand gravity, long-distance conduction as well other
environmental factors. Furthermore, wood formation in trees is very complex
and involves distinct physiological and anatomic processes that cannot be
addressed in the herbaceous model plant (Nieminen, Blomster, Helariutta, &
) such as perennial secondary growth,long life-span, cambium
dormancy, cambium ageing, juvenile and mature wood, heartwood formation.
In this review, we focus on recent findings on the regulation of wood
formation in angiosperm trees species, especially poplar and eucalypts,
highlighting conserved and distinct mechanisms with Arabidopsis. We first
provide illustrative examples of the emerging role of plant hormones in
the regulation of wood differentiation (including cell expansion and second-
ary wall deposition) and on the cross-talk between hormones. We then
survey the main transcription factors (TFs) from Populus and/or Eucalyptus
involved in the regulation of secondary cell wall formation and more
particularly those that have been recently functionally characterized in trees.
In the third part, we describe recent findings on the mechanisms regulating
the SW-regulators. We end discussing future outlines and challenges to fill
gaps in this exiting research field.
203Regulation of wood formation
2. Hormonal control of wood formation
It is not our intent here to cover the extensive literature on the hor-
monal control of cambium activity and cell specification by auxin, cytoki-
nin, gibberellins which were developed in other excellent reviews
(Bhalerao & Fischer, 2014;Mauriat et al., 2014;Milhinhos & Miguel,
2013;Nieminen, Robischon, Immanen, & Helariutta, 2012;Smet & De
Rybel, 2016;Sorce, Giovannelli, Sebastiani, & Anfodillo, 2013). We will
focus on the most recent and significant findings and especially those illus-
trating the role of auxin and its cross-talk with other hormones in initiating
the transcriptional programme of secondary xylem differentiation in trees.
Indeed, a recent publication described the spatial distribution of the plant
hormones auxin, gibberellin and cytokinin in poplar sections encompassing
the phloem, the vascular cambium and the xylem (Immanen et al., 2016).
They showed that each hormone exhibits distinct concentration maxima
with partially overlapping distribution profiles: auxin peaking in the cambial
zone, cytokinins in the developing phloem cells and gibberellin in expan-
ding xylem cells. This suggests that auxin interacts with other hormones
to contribute to subsequent cell fate decisions.
2.1 Reading the auxin gradient
In wood-forming tissue, auxin concentrations peak in the cambium and
decay rapidly towards the xylem and phloem (for review, see Bhalerao &
Fischer, 2014 and references therein, Immanen et al., 2016). The mecha-
nisms underlying the establishment of this gradient and how it could be
interpreted have been deeply discussed by Bhalerao and Fischer (2014).
The landmark work of Nilsson et al. (2008) showed that auxin-responsive
genes in wood-forming tissues of hybrid aspen respond dynamically to
changes in cellular auxin levels but the expression patterns of most of them
displayed limited correlation with the auxin concentration across this devel-
opmental zone. They also demonstrated that the perturbation of auxin sig-
nalling by overexpression of stabilized mutated version of the Aux/IAA,
PttIAA3, in transgenic aspen leads to a reduced cambial cell division while
radial extension of the cambial zone increases (Nilsson et al., 2008). This
suggests that auxin signalling not only promotes cambium proliferation
but also spatially restricts stem cell characteristics within the cambium area
(Bhalerao & Fischer, 2014). Notably, the PttIAA3-overexpressing trees also
produced smaller fibre and vessel cells than wild-type trees supporting a role
204 Eduardo L.O. Camargo et al.
of auxin not only in regulating the cell divisions that give rise to secondary
xylem cells but also in their subsequent expansion and development.
A possible interpretation of the auxin gradient proposed by Bhalerao and
Fischer (2014) is that high auxin concentrations are a signal for cell division,
intermediate levels may promote cell expansion and low levels may be read
out as a signal inducing the deposition of secondary cell walls (Bhalerao &
Fischer, 2014 and references therein). It is also worth noting that very
recently, Johnson et al. (2018) provided evidence that polar auxin transport
is important for vessel spatial patterning and size determination and by alter-
ing auxin transport, it is possible to shape the basic hydraulic properties of a
woody stem.
2.2 The interplay between auxin and the WOX-CLE-PXY
Recent work in Arabidopsis helped to unravel how auxin can regulate cam-
bial activity. Cambial cell division is promoted by the release of the phloem
RELATED41) which can bind its LRR-RLK receptor PXY (PHLOEM
INTERCALATED WITH XYLEM) in the cambium. Receptor signalling
then promotes cambial activity through the transcription factor WOX4
(WUSCHEL-related HOMEOBOX4 gene) (Ru
ˇka, Ursache, Heja
´tko, &
Helariutta, 2015). WOX4 mediates the auxin-dependent induction of cam-
bium activity and this process is dependent on PXY which is required
for a stable auxin-dependent WOX4 activity regulation (Suer, Agusti,
Sanchez, Schwarz, & Greb, 2011). Very recently, a breakthrough was
achieved by Brackmann et al. (2018) demonstrating the importance of
the spatial organization of the auxin signalling in the cambium. They
ARF5/MONOPTEROS are cambium regulators with different tissue-
specificities as well as distinct roles in cambium regulation. ARF3 and
ARF4 function redundantly as cambium activators. ARF5 acts by directly
attenuating the activity of the WOX4 gene, specifically in PXY-positive
cambium stem cells, in order to foster the transition from stem cells to dif-
ferentiated vascular cells (Brackmannetal.,2018).
Populus orthologs of CLE,PXY and WOX4 have similar expression
patterns to those in Arabidopsis suggesting that the CLE-PXY-WOX4
signalling pathway is conserved between Arabidopsis and woody plants
(Ursache, Nieminen, & Helariutta, 2013;Zhang, Nieminen, Serra, &
Helariutta, 2014; and references therein). Indeed, Etchells, Mishra,
205Regulation of wood formation
Kumar, Campbell, and Turner (2015) showed that an aspen receptor
kinase PttPXY and its peptide ligand PttCLE41 act to control a
multifunctional pathway that regulates both the rate of cambial cell divi-
sion and woody tissue organization. Whereas ectopic overexpression of
PttPXY and PttCLE41 genes in hybrid aspen resulted in vascular tissue
abnormalities and poor plant growth, their tissue-specific overexpression
generated trees that exhibited a two-fold increase in the rate of wood for-
mation, were taller, and possessed larger leaves compared to the controls.
More recently, Kucukoglu, Nilsson, Zheng, Chaabouni, and Nilsson
(2017) investigated the role of WOX4 and CLE41 orthologs in Populus
trees. They showed that PttWOX4 genes (PttWOX4a/b) were specifically
expressed in the vascular cambium during vegetative growth, but neither
after growth cessation nor during dormancy. In transgenic trees with
reduced expression levels of PttWOX4a/b, both periclinal and anticlinal
cambial cell divisions were reduced, causing a dramatic inhibition of sec-
ondary growth. This activity involves the positive regulation of
PttWOX4a/b through PttCLE41-related genes likely involving a regula-
tion by auxin. Based on their transcriptional data, the authors concluded
that a similar mechanism involving PttWOX4a/b,PttCLE41-related genes
and the gene PttPXYa regulates the identity and activity of the vascular cam-
bium in trees (Kucukoglu et al., 2017). The fine spatial organization of sig-
nalling events of auxin involving ARF5-dependent attenuation of WOX4
revealed in Arabidopsis (Brackmann et al., 2018) needs to be demonstrated
in trees.
2.3 The cross-talk between auxin and cytokinins stimulates
cambium activity
Cytokinins (CK) signalling is known to be important in the maintenance and
proliferation of cambial cells and in cambium cell specification (Bhalerao,
Fischer, & Turner, 2016;Milhinhos & Miguel, 2013; and references therein).
A reduction in cytokinin level by overexpression of a CK catabolic gene,
Arabidopsis CYTOKININ OXIDASE 2 (CKX2), in transgenic poplar leads
to a decrease in the number of cambium cells and concomitantly a reduced
stem diameter (Nieminen et al., 2008). On the other hand, aspen trees over-
expressing the AtIPT7 gene, encoding one key enzyme in the biosynthesis of
major bioactive CKs, displayed stimulated cambial cell division activity
resulting in dramatically increased production of the lignocellulosic trunk
206 Eduardo L.O. Camargo et al.
biomass. The elevation of the CK content led to an increase in cambial auxin
concentration, highlighting the interconnected nature of thesetwo hormonal
gradients to stimulate cambial activity (Immanen et al., 2016).
2.4 The cross-talk between auxin and gibberellins promotes
expansion of cambial derivatives
The role of gibberellin (GA) in wood development in trees is supported by
several lines of evidence (Mauriat et al., 2014;Milhinhos & Miguel, 2013;
and references therein). For instance, the overexpression of GIBBERELLIN
20-OXIDASE1 (GA20ox), a GA biosynthetic gene, in poplar results in
increased growth and xylem fibre length (Eriksson, Israelsson, Olsson, &
Moritz, 2000). Overexpression of poplar orthologs of AtGID1 (GIBBER-
ELLIN INSENSITIVE DWARF1) resulted in phenotypes similar to those
of the constitutive overexpressors of GA20ox, i.e., increases in xylogenesis
and cell elongation with the exception of increased fibre length (Mauriat &
Moritz, 2009). Noteworthy, the increase in xylogenesis in decapitated aspen
trees is stronger when adding both IAA and GA compared to what is
observed with adding only one hormone at a time (Bjorklund, Antti,
Uddestrand, Moritz, & Sundberg, 2007). Consistent with the alteration
of cambium proliferation observed in poplars affected in GA metabolism,
GA stimulates IAA transport and in turn, IAA stimulates GA biosynthesis
genes likely through Aux/IAA-ARF signalling elements. Strikingly, both
hormones shared a common transcriptomic signature including many tran-
scripts related to cell growth. These findings strongly support that these two
hormones play important roles in the post-meristematic expansion of cam-
bial derivatives (Bjorklund et al., 2007).
The gradual reduction in auxin concentration with increasing distance
from the cambium and concurrent increase concentration of active GA
may constitute a signal for cells to transition from an expansive phase to
one of maturation (Immanen et al., 2016). Building on the hypothesis that
the interplay between GA and IAA may act as a signal to initiate fibre dif-
ferentiation, Johnsson et al. (2018) tested if these hormones were able to reg-
ulate the expression of the top level regulators of xylem cell fate and SW
deposition, the secondary wall NAC (NAM/ATAF/CUC)-domain tran-
scription factors (see Section 3.1.1 and Table 1). Based on high spatial reso-
lution profiling in Populus (Sundelletal.,2017) and comparative phylogeny,
they first identified two clades of secondary wall NACs presenting distinct
207Regulation of wood formation
Table 1 Putative E. grandis and P. trichocarpa orthologs of Arabidopsis NAC domain proteins known to be involved in regulating secondary cell wall
NAC protein Arabidopsis
Putative Eucalyptus
ortholog (Hussey,
Saidi, Hefer, Myburg, &
Grima-Pettenati, 2015) Putative poplar co-orthologs References
B1/PtrNST1-1 (Potri.014G104800)
B2/PtrNST1-2 (Potri.002G178700)
Ohtani et al. (2011),Zhong, Lee,
and Ye (2010a, 2010b),Li et al.
(2012),Johnsson et al. (2018)
NST1 (ANAC043) EgrNAC49 PtVNS11/PtrWND1B/PtrSND1-A2/
PtrSND1-A2/PtrSND1-2 (Potri.001G448400)
A1/PtrSND1-1 (Potri.011G153300)
NST2 (ANAC066)
EgrNAC146 PtVNS01/PtrWND5A/PtrVND6-
C1/PtrVND3-1 (Potri.007G014400)
PtrVND3-2 (Potri.005G116800)
Lin et al. (2013),Ohtani et al. (2011),
Zhong et al. (2010a, 2010b),Li et al.
(2012),Johnsson et al. (2018),
Sundell et al. (2017)
VND3 (ANAC105)
VND4 (ANAC007)
VND5 (ANAC026)
EgrNAC50 PtVNS03/PtrWND4A/PtrVND6-
B2/PtrVND6-3 (Potri.001G120000)
B1/PtrVND6-4 (Potri.003G113000)
Ohtani et al. (2011),Zhong et al.
(2010a, 2010b),Li et al. (2012),
Johnsson et al. (2018)
VND6 (ANAC101) EgrNAC26 PtVNS05/PtrWND3A/PtrVND6-
A1/PtrVND6-1 (Potri.015G127400)
A2/PtrVND6-2 (Potri.012G126500)
Ohtani et al. (2011),Zhong et al.
(2010a, 2010b),Li et al. (2012),
Johnsson et al. (2018)
VND7 (ANAC030) EgrNAC75 PtVNS07/PtrWND6A/PtrVND7-1
Ohtani et al. (2011),Zhong et al.
(2010a, 2010b),Johnsson et al.
SND SND2 (ANAC073) EgrNAC170 PopNAC154/PtrSND2-2 (Potri.017G016700)
PopNAC156/PtrSND2-1 (Potri.007G135300)
Grant, Fujino, Beers, and Brunner
(2010),Johnsson et al. (2018)
SND3 (ANAC010) EgrNAC44
PopNAC157/PtrSND3-1 (Potri.004G049300)
PopNAC105/PtrSND3-2 (Potri.011G058400)
Grant et al. (2010),Johnsson et al.
XND1 (ANAC104) EgrNAC137
PopNAC118 (Potri.003G022800)
PopNAC122 (Potri.005G064100)
PopNAC128 (Potri.001G206900)
PopNAC129 (Potri.007G105000)
Grant et al. (2010),Johnsson et al.
VNI2 (ANAC083) EgrNAC122 Potri.015G102100 Johnsson et al. (2018)
expression profiles and containing in their promoters, both GA and IAA
response elements. Interestingly, they reported that those associated with fibre
and SW formation, the so-called “SND/NST”, were induced by GA treat-
ment and had a tendency to be repressed by auxin. On the other hand, both
GA and auxin could induce vessel-specific “VND” genes. These findings
were reflected at the anatomical level. In decapitated poplar stems, exogenous
auxin treatment reduced cell wall thickness while GA promotes SW deposi-
tion. High concentration of auxin in the cambial and expansion zones
represses fibre-specific NAC TFs and allows differentiation of xylem vessels.
When auxin concentration decreases and GA levels are high, all wood-
associated NACs are induced ( Johnsson et al., 2018). This supports a central
role of the IAA/GA cross-talk in determining cell fate possibly through the
control of SND/NST and VND master regulators and/or direct regulation
of SW biosynthesis ( Johnson et al., 2018). However, the underlying mech-
anisms of how auxin and GA coordinate expression of wood-associated NAC
TFs remain unclear although the presence of overlapping GA and auxin
responses elements in the promoter of some wood-associated NAC genes
could provide a way for the suggested IAA/GA cross-talk.
It is also worth noting that deregulation of a SND/NST member can, in
turn, affect auxin homeostasis. For instance, transformation of the PtSND2
activator into a strong repressor affects auxin biosynthesis, transport and sig-
nalling and, as a result, represses the normal growth and vascular develop-
ment of transgenic poplar plants (Wang et al., 2014). The promoters of
genes involved in auxin biosynthesis, transport and signalling contain SNBE
sites suggesting that they could be targets of NAC TFs ( Johnson et al., 2018).
2.5 Interaction between auxin and other hormones
Pioneer works on Zinnia mesophyll cell cultures demonstrated that bra-
ssinosteroids (BRs) are able to regulate xylem differentiation (Yamamoto
et al., 2007). In Arabidopsis shoots, Ibanes, Fabregas, Chory, and Cano-
Delgado (2009) showed that auxin polar transport coupled to BR signalling
is required to determine the radial pattern of vascular bundles. Recently, Jin
et al. (2017) have overexpressed one poplar ortholog of AtCYP85A2,
known to encode a bifunctional cytochrome P450 monooxygenase,
catalysing a final rate-limiting step in the BR-biosynthetic pathway. Over-
expression of PtCYP85A3 in poplar increased the endogenous BR levels
and significantly promoted growth, particularly xylem formation without
apparent alteration of SW properties ( Jin et al., 2017).
210 Eduardo L.O. Camargo et al.
The work of Agusti et al. (2011) revealed a role for strigolactone (SL)
signalling in the regulation of secondary growth conserved among species.
Exogenous application of artificial SL (GR24) on stems of Eucalyptus globulus
induced cambium division and Arabidopsis SL deficient mutants exhibited
less radial growth while presenting high IAA levels and signalling (Agusti
et al., 2011). The use of double mutants SL and IAA deficient suggested that
SL function predominantly downstream of auxin signalling that positively
regulates secondary growth.
2.6 Identifying new Aux/IAAs and ARFs as mediators of auxin
signalling in wood formation
Recognition of hormonal signals may require receptors with varying affin-
ities for the respective hormones, which, in the case of auxin, can be medi-
ated through differential expression of Aux/IAAs (Bhalerao & Fischer,
2014). Interestingly, in both poplar and Eucalyptus, analyses of xylem gene
networks revealed several Aux/IAAs as being tightly correlated with sec-
ondary wall TFs ( Johnson et al., 2018; R. Ployet et al., unpublished). With
exception of PtIAA3 (Nilsson et al., 2008) whose overexpression reduces
the width and the length for both fibre and vessels in transgenic lines in addi-
tion to a reduction of cambial cell division, none of the Aux/IAAs has been
yet proven to be involved in wood formation. In Arabidopsis, ARFs 3 and 4
were recently pointed out as general promoters of cambial activity whereas
ARF5 has a more specific role in attenuating the activity of WOX4 to
stimulate differentiation (Brackmann et al., 2018). With the objective of iden-
tifying auxin signalling mediators involved in wood formation, a genome-
wide identification of the ARFs and Aux/IAAs gene families was performed
in Eucalyptus grandis (Yu et al., 2015, 2014). While the E. grandis has one of the
largest proportion of tandem duplicated genes (34%; Myburg et al., 2014),
these two families which contain 17 ARFs and 24 Aux/IAAs members,
respectively, are not affected at all by tandem duplication. They contain much
less genes than poplar (35 ARFs, 39 Aux/IAAs) and strikingly even less than
Arabidopsis (23 ARFs and 29 Aux/IAAs). A combination of comparative phy-
logenetic analysis and large-scale gene expression profiling enabled the iden-
tification of several candidates. Functional characterization of EgrIAA4 by
overexpressing a stabilized version in Arabidopsis dramatically impeded plant
growth and fertility and induced auxin-insensitive phenotypes. The lignified
secondary walls of the interfascicular fibres appeared very late, whereas those
of the xylary fibres were virtually undetectable, suggesting that EgrIAA4 may
play crucial roles in fibre development and secondary cell wall deposition
211Regulation of wood formation
(Yu et al., 2015). EgIAA20 and EgIAA9 had a similar inhibiting effects on
fibres although the later also triggered prominent vessel formation in second-
ary xylem (H. Cassan-Wang, unpublished).
2.7 Role of ethylene in xylem cell expansion and maturation
The exogenous application of ethylene stimulates cambium activity but not
in the ethylene-insensitive transgenic poplar obtained by overexpressing a
dominant negative Arabidopsis ethylene receptor ETR1 (Love et al.,
2009). Consistent with these observations, the overexpression of ethylene
biosynthesis gene PttACO1 (ACC oxidase) stimulates cambium prolifera-
tion in Populus. The authors also showed that ethylene is at the origin of
the eccentric cambial activity which gives rise to reaction wood formation
in response to leaning.
More recently, Seyfferth et al. (2018) started to elucidate the ethylene sig-
nalling pathway in Populus trees and identified several putative downstream
targets. In silico analysis of the AspWood transcriptome database (http://, which covers all stages of secondary growth in
aspen stems, revealed that the ethylene precursor 1-aminocyclopropane-
1-carboxylic acid (ACC) is synthesized during xylem expansion and cell
maturation. More interestingly, ethylene-mediated transcriptional repro-
gramming occurs during all stages of secondary growth. The authors also iden-
tified new putative regulatory hub genes like EIN3D (ETHYLENE
which, for most of them, were connected for the first time to wood formation
(Seyfferth et al., 2018).
3. Main actors of the SW regulatory network:
NACs and MYBs
The deposition of lignified SWs is a major step in the differentiation of
secondary xylem. It is of prime importance for wood for fulfilling its essential
roles in trees. It is also determinant for wood end-uses by mankind. In the
last two decades, tremendous progress had been made in our understanding
of the transcriptional regulation of this strictly coordinated spatiotemporal
process thanks to pioneer work using the Zinnia and Arabidopsis trans-
differentiation systems as well as using reverse and forward genetics in the
model plant Arabidopsis (for reviews, see Demura & Fukuda, 2007;
Grima-Pettenati, Soler, Camargo, & Wang, 2012;Hussey, Mizrachi,
212 Eduardo L.O. Camargo et al.
Creux, & Myburg, 2013;Nakano, Yamaguchi, Endo, Rejab, & Ohtani,
2015;Schuetz et al., 2013;Umezawa, 2009;Wang & Dixon, 2011;
Zhao & Dixon, 2011;Zhong, Lee, et al., 2011;Zhong, McCarthy,
Lee, & Ye, 2011;Zhong & Ye, 2009, 2015).
In Arabidopsis, a complex hierarchical regulatory network has been pro-
posed to control SW deposition in which the transcription factors NAC
(NAM/ATAF/CUC) and MYB (MYeloBlastosis) act as first- and
second-level master switches, respectively, to regulate a battery of down-
stream transcription factors and secondary cell wall biosynthesis genes
(Schuetz et al., 2013;Wang & Dixon, 2011). Strong evidence also supports
an Arabidopsis-like transcriptional cascade in woody angiosperm species
(Hussey et al., 2013;Zhong, Lee, et al., 2011;Zhong, McCarthy, et al.,
2011;Zhong & Ye, 2015). However, the growing number of functional
genomic studies performed in trees highlighted differences in the regulation
system for SW thickening between annual herbaceous plants and woody
angiosperms. Our intent is to highlight the most significant and recent results
in this rapidly evolving area, which illustrate the high complexity of the tran-
scriptional regulation of the SW in trees. We will review recent progress on
the functional characterizations of poplar and eucalypts NACs and MYBs
and of the emerging mechanisms regulating their activities.
3.1 Wood-associated NAC domain transcription factors
The discovery of the NAC-domain TF genes as the key players regulating
the complex transcriptional network leading to wall-thickening cell differ-
entiation represents a milestone in our understanding of the regulation of
SW (Kubo et al., 2005).
3.1.1 Top-level NAC master regulators
In Arabidopsis, there is clear separation in the expression patterns of the seven
VNDs (Vascular-Related NAC Domain: AtVND1 to AtVND7) which are
preferentially expressed in xylem vessels and the five NAC genes named
either NSTs (NAC Secondary Wall thickening promoting factor) or SNDs
(Secondary Wall-Associated NAC Domain: AtNST1, AtNST2 and
AtNST3/AtSND1/ANACO12, SND2 and SND3) which are mostly
expressed in interfascicular fibres. AtVNDs induce differentiation of vessels
whereas NSTs/SNDs regulate SW deposition in fibres (Mitsuda et al., 2007;
Mitsuda, Seki, Shinozaki, & Ohme-Takagi, 2005;Yamaguchi, Kubo,
Fukuda, & Demura, 2008;Zhong, Demura, & Ye, 2006).
213Regulation of wood formation
Overexpression of AtVND6 or VND7 was shown to induce xylem ves-
sel transdifferentiation both in Arabidopsis and in poplar (Kubo et al., 2005;
Yamaguchi et al., 2010), suggesting that the molecular mechanism of xylem
vessel differentiation is, at least partially, conserved between these two
The same poplar genes were named differently depending on the authors
(see Table 1) as WNDs (for wood-associated NAC domain transcription fac-
tors, Zhong et al., 2010a, 2010b) or VNSs (for VND, NST/SND-,
SOMBRERO-related proteins; Ohtani et al., 2011) or PtrSNDs/PtrVNDs
(Johnsson et al., 2018;Li et al., 2012). To help the reader finding his way in
this confusing nomenclature, we reported in Table 1, the short names of the
putative co-orthologs of the Arabidopsis genes in poplar and eucalypts, given
by different research groups. The poplar orthologs of the VNDs and/or
NSTs/SNDs genes could complement the SW defects of the fibres in the
double nst1/(snd1/nst3/anac012)Arabidopsis mutant (Zhong & Ye, 2010).
However, surprisingly only PtrWND2B and PtrWND6B were able to
induce ectopic deposition of SW when overexpressed in Arabidopsis
(Zhong et al., 2010a, 2010b). To explain the fact that other VNDs or
NST/SNDs members could complement the nst1/snd1 mutant but are
not capable of inducing ectopic expression of SWs, a likely hypothesis is that
they need to cooperate with co-factors or other TFs which are only present
in cells programmed to be sclerified.
It was initially reported that the fibre- or vessel-specific expression
occurring in Arabidopsis was not occurring in poplar where all WNDs/
VNS (both VND and NST/SND) were expressed in both developing ves-
sels and fibres as well as in xylem ray parenchyma cells. The clear separation
of the expression patterns of VND and NST/SND groups in Arabidopsis did
not seem to be extensively shared with other plant species including poplar,
rice, and maize (Nakano et al., 2015;Ohtani et al., 2011;Zhong, Lee, et al.,
2011;Zhong et al., 2010a, 2010b;Zhong, McCarthy, et al., 2011). How-
ever, the recent high spatial-resolution RNA sequencing data spanning the
secondary phloem, vascular cambium, and wood-forming tissues of Populus
tremula (Johnsson et al., 2018;Sundell et al., 2017) provided new clues about
the expression patterns of the genes of the VNDs and NSTs/SNDs clades
which are more complex and subtle than previously thought. Considering
cell distance from cambium as a proxy for cell age, Johnsson et al. (2018)
showed that VND6-orthologs are induced in recently divided cambial
xylem initials, while SND1/NST1 orthologs are expressed in early xylem
expansion zone. The expression patterns within the paralogous pairs of
214 Eduardo L.O. Camargo et al.
PttSND1,PttNST1, and PttVND6 are highly similar whereas divergent
expression profiles were observed within the PttVND3 and PttVND7 pairs.
Both of the PttVND7 paralogs are expressed in primary xylem. PttVND7-1
is induced in the end of the maturation/programmed cell death zone indi-
cating neofunctionalization of this paralog whereas PttVND7-2 is not
detectable in the secondary xylem ( Johnsson et al., 2018). These authors fur-
ther investigated the differences between the PtrSND and PtrVND clades by
generating a co-expression network. They showed that PtrVND6 orthologs
together with PtrVND3-2 formed a cluster connected to the bulk of pectin
and xyloglucan biosynthetic genes as well as three primary wall-associated
cellulose synthases PtrCESAs.PtrSND1 and PtrNST1 orthologs were most
closely connected to secondary wall-associated PtrCESAs.
Using PtrWND2B/6B as tools, Zhong, McCarthy, et al. (2011) have
uncovered asuite of up-regulated TFs, many of which were not reported
previously. They further showed that PtrWNDs/VNS bind directly to
SNBE sites in the promoters of their target genes like Arabidopsis SND1
(Zhong, Lee, et al., 2011;Zhong, McCarthy, et al., 2011).
More work is needed to decipher the target genes of the different mem-
bers of the two clades (VND and NST/SND) since indirect evidence sup-
ports the occurrence of a divergence in target genes between clades. Indeed,
the identification of co-varying neighbouring genes showed that if there are
some common genes shared in the neighbourhoods of members of the same
clade, either VND or NST/SND, there is no overlap between members
of the two distinct clades ( Johnsson et al., 2018). Using Populus trichocarpa
protoplasts from stem-differentiating xylem, Lin et al. (2013) have started
to describe the SND/VND-directed functional hierarchical genetic regula-
tory network (hGRN) for wood formation. They monitored genome-
wide PtrSND1-B1 (PtVNS9/PtrWND2A)-induced gene transactivation
responses and used a novel computational method for constructing a hier-
archical TF-DNA network involving 76 direct targets, including 8 TFs and
61 enzyme-coding genes previously unidentified as targets (Lin et al., 2013).
3.1.2 Second level NAC proteins involved in wood formation:
Downstream the top-level NAC regulators, AtSND2 and AtSND3,
expressed in SW-associated tissues, are direct target of AtSND1 and function
as second level regulators for SW formation (Zhong, Lee, Zhou,
McCarthy, & Ye, 2008). Overexpression of AtSND2 in Arabidopsis stems pro-
duced a specific increase in xylem fibre wall thickening (Zhong et al., 2008)
215Regulation of wood formation
whereas overexpression of one of the four putative SND2 co-orthologs in pop-
lar, PopNAC154, resulted in a decrease in height and an increase in the propor-
tion of bark to xylem in poplar trees, with no perceptible effect on SW thickness
(Grant et al., 2010). This apparent discrepancy between the SND2 over-
expression phenotype in Arabidopsis and poplar was initially interpreted as illus-
trating differences in the regulatory function of SND2 orthologs between
herbaceous and woody plants. However, several lines of evidence suggest that
it is more complex than only reflecting species differences. First, overexpression
of AtSND2 in Eucalyptus increased the thickness of the SW in fibre cells (Hussey
et al., 2011). Second, overexpression of a dominant repressor form of a SND2
poplar ortholog (PtSND2-SDRX) repressed wood formation and xylem fibres
thickness in transgenic poplar lines (Wang et al., 2013). It should be noted that
Eucalyptus contains one single ortholog of AtSND2 (EgrNAC170,Hussey
et al., 2015) whereas poplar has four co-orthologs that may have undergone
sub/neofunctionalization. Third, excessive levels of AtSND2 overexpression
have a negative effect on interfascicular fibres SW deposition in Arabidopsis
(Hussey et al., 2011). The authors postulated that SND2 overexpression could
increase SW deposition within a limited range of overexpression levels, possibly
relying on the abundance of additional regulator proteins. The mode of regu-
lation of SND2 seems complex and requires further investigation.
Recently Johnsson et al. (2018) showed that poplar orthologs of
AtSND2 and AtSND3 all peak into the xylem maturation zone. While both
PtrSND2 orthologs cluster with the PtrSND1 and PtrNST1 orthologs and
are closely connected to SW PtrCESAs. The PtrSND3 orthologs sub-cluster
distinctly from the others together with primary wall PtrCESAs and other
cell wall biosynthetic genes. Strikingly, PtrSND3 orthologs were negatively
correlated with the vast majority of the genes in their cluster.
Given the severe growth phenotypes observed in poplar lines either
overexpressing PopNAC154 (Grant et al., 2010) or its dominant repressive
form (Wang et al., 2013) and the fact that PopNAC154 was down-regulated
in tension wood (Andersson-Gunnera
˚s et al., 2006;Grant et al., 2010). Jervis
et al. (2014) performed a metabolomic study in field-grown poplar over-
expressing PopNAC154. They observed a substantial increase in arginine
content which could be a marker for earlier growth cessation suggesting that
PopNAC154 could also play a role in senescence/dormancy-associated pro-
cesses. Wang, Li, et al. (2014) and Wang, Tang, et al. (2014) further inves-
tigated the phenotypes (reduced growth and xylem formation) caused by
dominant repression of PtSND2 (PopNAC154), and found a reduction of
auxin biosynthesis, transport and signalling in these transgenic poplar lines.
216 Eduardo L.O. Camargo et al.
3.1.3 Other NAC Proteins XND1
XYLEM NAC DOMAIN 1 (XND1) is a negative regulator of SW and
programmed cell death in Arabidopsis (Zhao, Avci, Grant, Haigler, &
Beers, 2008). Its overexpression results in extreme dwarfism associated with
the lack of SW synthesis and programmed cell death in xylem vessels. Poplar
has four XND1 co-orthologs PopNAC118,PopNAC122,PopNAC128,
PopNAC129, each of them exhibiting distinct expression patterns. The
overexpression of PopNAC122 or PopNAC129 in Arabidopsis phenocopied
AtXND1 overexpression in Arabidopsis (Grant et al., 2010). When AtXND1
was overexpressed in poplar vessel diameter and number, plant stature and
leaf size were reduced in severely affected lines. The effects of XND1 over-
expression in poplar did not precisely phenocopy those observed in
Arabidopsis but in both genetic backgrounds overexpression of XND1 was
associated with reduced plant size and inhibition of differentiation of a subset
of vascular cell types.
3.2 SW-associated MYB transcription factors
3.2.1 Second-level master regulators
In Arabidopsis,AtMYB46/83 are considered as the unique second-level mas-
ter regulators since they are the direct targets of top-level VND and
NST/SND master switches and are able to activate the promoters of all
the three major SW polymers (i.e. cellulose, hemicelluloses and lignins).
In poplar, the AtMYB46/83 co-orthologs, i.e., PtrMYB2,PtrMYB3,
PtrMYB20, and PtrMYB21, are direct targets of PtWND2B/6B (Zhong,
McCarthy, et al., 2011). As their Arabidopsis counterparts, they are also capa-
ble of activating the biosynthesis pathways of cellulose, xylan, and lignin,
leading to ectopic SW deposition when overexpressed in Arabidopsis and
poplar. Their dominant repression results in a reduction of SW thickening
in transgenic poplar wood (McCarthy et al., 2010;Zhong, McCarthy,
Haghighat, & Ye, 2013). EgMYB2 from Eucalyptus (Goicoechea et al.,
2005) is also an ortholog of AMYB46/83 since it is able to activate the entire
secondary wall biosynthesis programme when overexpressed and can com-
plement the Arabidopsis mutant atmyb46/83 (Zhong et al., 2010a).
Interestingly, overexpression of BplMYB46 from Betula platyphylla a
close ortholog of PtrMYB2/3/20/21 not only increases lignin deposition,
SW thickness and the expression of SW biosynthesis genes but also improves
salt and osmotic tolerance in transgenic birch plants (Guo et al., 2017). It
should be noted that its overexpression induces overexpression of an ortho-
log of AtMYB52 known to confer ABA hypersensitivity and drought
217Regulation of wood formation
tolerance in Arabidopsis (Park, Kang, & Kim, 2011). BplMYB46 is one of the
first examples highlighting the cross-talk between the abiotic stress and sec-
ondary cell wall biosynthesis pathways in trees (Guo et al., 2017).
One important difference between Arabidopsis and poplar is that other
targets of PtWND2B/6B than the orthologs of AtMYB46/83 (PtrMYB 2,
3,20 and 21) are capable of inducing the whole SW transcriptional pro-
gramme and may function as master switches (Zhong, McCarthy, et al.,
2011). Among those, PtrMYB18 (ortholog of AtMYB20/43), PtrMYB75/
92/125/199 (co-orthologs of AtMYB42/85), PtrMYB10/128 (co-
orthologs of AtMYB103), PtrMYB74 and PtrMYB121 can activate the pro-
moter activities of several biosynthesis genes for cellulose, xylan, and lignin
(Zhong, Lee, et al., 2011;Zhong, McCarthy, et al., 2011). PtrMYB74 and
PtrMYB121 were among the 13 targets induced by PtWND2B, for which
no ortholog of Arabidopsis was previously shown to be involved in the reg-
ulation of SW biosynthesis. It is possible that additional master switches have
been recruited in poplar to sustain a robust expression of secondary wall bio-
synthesis genes during wood formation, which requires the deposition of a
massive amount of secondary wall components. Another hypothesis is that
these genes have specific cell localization and/or have diversified their func-
tions for being involved in responses to stresses. Examples of such a diver-
sification are emerging. BplMYB46 (Guo et al., 2017) and PtoMYB170
(Xu et al., 2017, see below) are both at the cross-road between SW forma-
tion and response to drought.
Among the poplar wood-associated MYB having diversified their tran-
scriptional regulatory activities from those of their Arabidopsis counterparts is
PtrMYB128 (an ortholog of AtMYB103). PtrMYB128 is capable to activate
the promoters of the biosynthetic genes for all three secondary wall compo-
nents in transient transactivation assays (Zhong, Lee, et al., 2011;Zhong,
McCarthy, et al., 2011). AtMYB103 was first shown to preferentially induce
the expression of genes for the biosynthesis of cellulose but not xylan and
lignin (Zhong et al., 2008). However, the characterization of two
myb103 Arabidopsis T-DNA insertion mutants revealed that the main mod-
ification at the SW level was a change in lignin monomeric composition
(decrease in the S/G ratio) in line with FERULATE 5 HYDROXYLASE
(F5H) being the main target of AtMYB103 (
Ohman et al., 2013).
PtrMYB152 and its paralog PtrMYB18 are orthologs of the Arabidopsis
paralogs AtMYB43 and AtMYB20, respectively (Wang, Li, et al., 2014;
Wang, Tang, et al., 2014). Both AtMYB43 and AtMYB20 were identified
as targets of AtSND1 but their functions in SW biosynthesis programme
218 Eduardo L.O. Camargo et al.
have not been characterized. PtrMYB18 was identified as up-regulated in
response to PtrWND2B, but not PtrMYB152. When overexpressed in Ara-
bidopsis,PtrMYB152 increased secondary cell wall thickness, likely caused by
increased lignification. Accordingly, elevated expression of genes encoding
sets of enzymes in secondary wall biosynthesis was observed (Wang, Li,
et al., 2014;Wang, Tang, et al., 2014).
3.2.2 Lignin-specific MYBs
In Arabidopsis, three lignin specific MYB TFs have been identified:
AtMYB58, AtMYB63 and AtMYB85 (Zhong & Ye, 2009). PtrMYB28
is able to activate specifically lignin biosynthesis genes as its orthologs
AtMYB58/MYB63 (Zhong & Ye, 2009). Overexpression of PtoMYB92,
an ortholog of MYB85/MYB42 resulted in an increase in SW thickness in
stems and ectopic deposition of lignin in leaves. PtoMYB92 specifically acti-
vates the expression of lignin biosynthetic genes. Thus, PtoMYB92 is
involved in the regulation of SW formation in poplar by controlling the bio-
synthesis of monolignols as its Arabidopsis counterpart (Li et al., 2015).
The respective roles of the two paralogs PtoMYB170 and PtoMYB216
(co-orthologs of AtMYB61) were examined by Tian et al. (2013) and Xu
et al. (2017). Using overexpression in poplar, they first showed that
PtoMYB216 and PtoMYB170 have conserved functions in regulating lignin
biosynthesis. Both TFs specifically activate the expression of the upstream
genes in the lignin biosynthetic pathway which results in ectopic deposition
of lignin in cells that are normally non-lignified (Tian et al., 2013). How-
ever, the CRISPR/Cas9 mutant of PtoMYB170 displays pendent stem phe-
notype resulting from reduced lignin deposition and more flexible SW of
xylem fibres. This strong phenotype suggests that the functional redundancy
between PtoMYB216 and PtoMYB170 is weak because PtoMYB216 can-
not replace the function of PtoMYB170 in lignin deposition (Xu et al.,
2017). Interestingly, heterologous expression of PtoMYB170 in transgenic
Arabidopsis enhanced stomatal closure in the dark and resulted in drought
tolerance of the transgenic plants through reduced water loss. PtoMYB170
was specifically expressed in guard cells of transgenic Arabidopsis while
PtoMYB216 was not. These findings illustrate of a neofunctionalization of
one of the two paralogs which acquired a new function in enhancing
drought tolerance. After BplMYB46 (Guo et al., 2017) PtoMYB170 is
the second example of tree SW-TF able to integrate both developmental
and stress signals.
219Regulation of wood formation
3.2.3 Negative regulators
SW formation not only involves transcriptional activators but also entails
transcriptional repressors. Eucalyptus EgMYB1 represses the expression of
secondary wall biosynthesis genes and inhibits secondary wall thickening
in fibres when overexpressed in Arabidopsis and poplar, suggesting that it
is a master transcriptional repressor of secondary wall formation (Legay
et al., 2007, 2010). Although, EgMYB1 is a close ortholog of AtMYB4, their
respective functions are different since AtMYB4 has never been reported to
regulate SW synthesis but instead it is known to regulate sinapate esters accu-
mulation through its direct target C4H (cinnamate 4-hydroxylase,
Jin et al., 2000).
The two EgMYB1’s orthologs in Populus have been functionally charac-
terized in independent studies. PdMYB221 was overexpressed in Arabidopsis
(Tang et al., 2015) and PtoMYB156 was overexpressed and down-regulated
by genome editing in poplar (Yang et al., 2017). Both genes were shown to
negatively regulate the secondary wall thicknesses of xylem fibres and the
content of cellulose, lignin and hemicelluloses but PtoMYB156 was also
shown to repress phenylpropanoid biosynthesis genes, leading to a reduction
in the amounts of total phenolic and flavonoid compounds (Yang et al.,
2017). Whether PdMYB221 also regulates genes of the phenylpropanoid
is still to be determined.
3.3 New NAC and MYB candidates putatively involved in the
control of wood formation in trees
The availability of the E. grandis genome, the second hardwood forest tree
whose genome was sequenced (Myburg et al., 2014), enabled to perform
comparative phylogeny studies with woody and nonwoody species.
Although the E. grandis genome has the very high number of tandem dupli-
cated genes (33%, Myburg et al., 2014), Eucalyptus orthologs of the main
NAC and R2R3-MYBs controlling secondary cell wall formation and lig-
nin biosynthesis are, in general, not duplicated. For instance, there are single
putative Eucalyptus orthologs of Arabidopsis fibre-associated SND1, SND2
and NST1, vessel-VND6, VND7, MYB46/83 (Hussey et al., 2015;Soler
et al., 2015). This contrasts to poplar who underwent recent whole genome
duplication (Tuskan et al., 2006) and has in general two duplicated orthologs
of one Arabidopsis SW main regulatory gene (Tables 1 and 2). In both NAC
and MYB families, there are clades exhibiting a high number of tandem
duplicated genes. Among those, the “woody-expanded” showing an expan-
sion of members from woody species and the “woody-preferential”
220 Eduardo L.O. Camargo et al.
Table 2 MYB transcription factors from angiosperm trees involved in SW formation.
species TFs Gene ID
Target genes
ReferencesLignin Cellulose Xylan
BplMYB46 KP711284 AtMYB46 BplPAL, Bpl4CL,
BplCesA BplFRA,
Guo et al.
EgMYB1 CAE09058 AtMYB4 EgCCR, EgCAD2, PtrPAL2,
Ptr4CL3, PtrCOMT2,
PtrHCT1, PtrCCR2,
PtrCAD1, PtrC3H3,
PtrC4H1, PtrC4H2
PtrCesA1,2,3 PtrFRA8,
Legay et al.
(2007, 2010)
EgMYB2 AJ576023 AtMYB46 NtPAL, NtC4H, Nt4CL,
et al. (2005)
EgMYB88 KX470407 NO PttPAL, Ptt4CL, PttCAld5H Soler et al.
PdMYB221 Potri.004G174400.1 AtMYB4 PdCOMT2, PdCCR1 PdCesA7/8 PdGT47C Tang et al.
PtrMYB2 Potri.001G258700 AtMYB46/83 At4CL1, AtCCoAOMT,
PtrCesA4,8,17 PtrGT43B,
et al. (2010),
Zhong et al.
PtrMYB3 Potri.001G267300 AtMYB46/83 PtrCOMT1,
PtrCesA4,8,17 PtrGT43B,
et al. (2010),
Zhong et al.
Table 2 MYB transcription factors from angiosperm trees involved in SW formation.contd
species TFs Gene ID
Target genes
ReferencesLignin Cellulose Xylan
PtrMYB20 Potri.009G061500 AtMYB46/83 PtrCOMT1,
PtrCesA4,8,17 PtrGT43B,
et al. (2010),
Zhong et al.
PtrMYB21 Potri.009G053900 AtMYB46/83 PtrCOMT1,
PtrCesA4,8,17 PtrGT43B,
et al. (2010),
Zhong et al.
PtrMYB152 Potri.017G130300 AtMYB43/20 At4CL1, AtHCT, AtC3H1,
PtrPAL4, Pt4CL3, Ptr4CL5,
PtrC4H2, PtrCCoAOMT1,
PtrCCR2, PtrHCT1
PtrCesA2B,3A PtrGT43B,D Li et al. (2014),
Wang, Li, et al.
Tang, et al.
PtrMYB28 XM_002307154 AtMYB58/63 At4CL Zhong and Ye
PtoMYB92 KP710214 AtMYB42/85 PtoCCOAMT1, PtoCCR2,
Li et al. (2015)
PtoMYB170 KY114929 AtMYB61 PtoCCR2,
PtoCOMT, PtoC4H2
Xu et al.
PtoMYB216 JQ801749 AtMYB61 PtoPAL4, Pto4CL5,
PtoC4H2, PtoC3H3
PtoCesA3A PtrGT43B,D Tian et al.
containing neither Arabidopsis nor monocot genes. Combining comparative
phylogeny to large scale expression profiling, enabled to point out new
NAC and MYB candidate putatively involved in the control of wood for-
mation in trees (Hussey et al., 2015;Soler et al., 2015). Of particular interest
are members expressed in vascular cambium and/or differentiating xylem
with no ortholog gene in Arabidopsis. Among these candidates, EgMYB88
which belongs to one woody subgroups and is highly and preferentially
expressed in vascular cambium was functionally characterized in poplar
(Soler et al., 2016). The results suggest that EgMYB88 is an activator of
the biosynthesis of some phenylpropanoid-derived secondary metabolites
including lignin in cambium and in the first layers of differentiating xylem.
4. Post-transcriptional and post-translational
regulations of the SW regulators
4.1 Alternative splicing regulates cell wall thickening
Post-transcriptional regulation plays an important role in modulating
VNS/WND activity. A major breakthrough was achieved by Li et al.
(2012) who reported the discovery of a stem-differentiating xylem (SDX)-
specific alternative SND1 splice variant, PtrSND1-A2
negative of SND1 transcriptional network genes in poplar. PtrSND1-A2
which derives from PtrSND1-A2 (also called PtrWND1B/PtrVNS11) lacks
DNA binding and transactivation abilities but retains dimerization capabil-
ity. PtrSND1-A2 and PtrSND1-A2
can form heterodimers with other
PtrSND members. PtrSND1-A2
which is localized exclusively in cyto-
plasm is translocated into the nucleus exclusively as a heterodimeric partner
with full-size PtrSND1s, where it represses the expression of the PtrSND1
member genes and their target gene PtrMYB021. This was the first report in
plants of the repression of the autoregulation of a TF family by its only splice
variant. Zhao, Sun, Xu, Zhang, and Li (2014) further showed that
PtrSND1-A2 overexpression enhanced fibre cell wall thickening, while
overexpression of PtrWND1B-l (PtrSND1-A2
) repressed fibre cell wall
thickening. Interestingly, its ortholog in Eucalyptus (Eugr.E01053) also
undergoes a form with a retention of intron 2 whereas such event does
not exist in Arabidopsis.
Very recently, Lin et al. (2017) reported the discovery of another splice
variant, PtrVND6-C1
family derived from PtrVND6-C1 (PtVNS01/
PtrWND5A). It suppresses the protein functions of all PtrVND6 family
223Regulation of wood formation
members. PtrVND6-C1
also suppresses the expression of all PtrSND1
members, including PtrSND1-A2, demonstrating that PtrVND6-C1
is the previously unidentified regulator of PtrSND1-A2. Similarly,
cannot suppress the expression of its cognate transcription
factor, PtrVND6-C1. PtrVND6-C1 is suppressed by PtrSND1-A2
. Both
and PtrSND1-A2
cannot suppress their cognate tran-
scription factors but can suppress all members of the other family, indicating
that the splice variants from the PtrVND6 and PtrSND1 family may exert
reciprocal cross-regulation for complete transcriptional regulation of these
two families in wood formation, providing a higher level of regulation to
maintain homeostasis for plants to avoid abnormal growth and development.
The importance of alternative splicing (AS) in the regulation of wood for-
mation deserves more attention since 36% of wood-expressed genes were
reported to be alternatively spliced in the xylem transcriptome in
P. trichocarpa (Baoetal.,2013). Xu et al. (2014) found that 28.3% and
20.7% of the highly expressed transcripts were affected by AS events in devel-
oping xylem of Populus and Eucalyptus, respectively. Around 25% of the AS
events may cause protein domain modification. About 30% of AS-occurring
genes were putative orthologs and 71 conserved AS events were identified in
the two species (Xu et al., 2014).
4.2 Proteinprotein interactions
Despite the importance of combinatorial control in transcriptional regulation
in plants, there are only a handful of examples of the involvement of
proteinprotein interactions in the network controlling secondary cell wall
formation. With the exception of the work on PtrVND and SND/NST
homo- or heterodimerization (Li et al., 2012;Shi et al., 2017)mostofthe
studies were performed in A. thaliana (Bhargava et al., 2013;Li et al.,
2012;Liu & Douglas, 2015;Liu et al., 2014;Yamaguchi et al., 2010). How-
ever, recently Soler et al. (2016) found that the Eucalyptus transcription factor
EgMYB1, which is known to repress lignin biosynthesis, interacts specifically
with a linker histone variant, EgH1.3. This interaction enhances the repres-
sion of EgMYB1’s target genes, strongly limiting the amount of lignin depos-
ited in xylem cell walls. The expression profiles of EgMYB1 and EgH1.3
overlap in xylem cells at early stages of their differentiation as well as in mature
parenchymatous xylem cells, which have no or only thin lignified secondary
cell walls. This suggests that a complex between EgMYB1 and EgH1.3 inte-
grates developmental signals to prevent premature or inappropriate lignifica-
tion of secondary cell walls, providing a mechanism to fine-tune the
differentiation of xylem cells in time and space (Soler et al., 2017).
224 Eduardo L.O. Camargo et al.
With the aim of deciphering the Populus wood interactome, Petzold
et al. (2017) have used yeast-two-hybrid and discovered 165 novel
proteinprotein interactions and connected networks for 162 distinct
genes. Some of these interactions were previously reported but most were
novel and relevant to wood formation since they involved proteins known
to be involved in SW synthesis (Petzold et al., 2017). This work will pro-
vide an excellent framework to further functionally characterize relevant
5. Concluding remarks
Within the last decade, and particularly within the 5 last years, our
understanding of the transcriptional regulation of wood formation in angio-
sperm trees has substantially progressed. The increasing number of func-
tional characterizations of trees SW-associated TFs revealed that
Arabidopsis has been instrumental in deciphering conserved mechanisms
involved in secondary xylem differentiation. However, substantial differ-
ences exist between trees and the model plant and we cannot further rely
on simple extrapolations to decipher the transcriptional network underlying
wood formation in trees given its higher level of complexity.
A growing number of examples illustrate the diversification of functions
of tree wood-associated TFs as compared to their putative orthologs in
Arabidopsis (Legayetal.,2010;Nakano et al., 2015;Zhong, McCarthy,
et al., 2011). Most of the functional studies have focussed on potential orthologs
of Arabidopsis genes and few investigated the function of TFs absent from the
Arabidopsis genome (Soler et al., 2015). Those expressed preferentially in vas-
cular cambium or differentiating xylem deserve more attention since they likely
play specific roles in trees (Husseyetal.,2015;Soler et al., 2015).
The overexpression strategy has been and still very useful but showed its
limits in deciphering the function of wood-associated TFs. An illustrative
example is AtMYB103 whose function determined thanks to Arabidopsis
T-DNA mutants (
Ohman et al., 2013) was shown to be different from that
deduced from overexpressors (Zhong, Lee, et al., 2011;Zhong, McCarthy,
et al., 2011). Implementation of genome editing technologies in poplar (e.g.
CRISPR-Cas9) provides a means to engineer strong loss-of-function or null
alleles, thereby complementing the lack of mutant in tree species. The
powerfulness of this approach was recently highlighted in deciphering the
function of two paralogs (PtoMYB126 and PtoMYB170) from Populus
tomentosa (Xu et al., 2017). As an alternative to poplar transformation which
is still a long process as compared to Arabidopsis, “Induced somatic sector
225Regulation of wood formation
analysis” (ISSA) is a rapid method implemented both in poplar and Eucalyptus,
which was successfully used by Hussey et al. (2011) to functionally character-
ize SND2 in Eucalyptus. This method is particularly adapted to study cambium
development dynamics in trees (Bossinger & Spokevicius, 2018). Another
alternative approach is to use Eucalyptus transgenic hairy roots, a versatile
and rapid system to dissect the function of genes involved in secondary wall
biosynthesis (Plasencia et al., 2016) and in its regulation (Soler et al., 2016).
One exiting outcome from the recent characterization of wood-
associated TFs involved in SW is their role in integrating environmental
signals leading to increased tolerance to abiotic stresses, (i.e. drought and
salinity stresses; Guo et al., 2017;Xu et al., 2017). It is likely that more exam-
ples will emerge in the near future as it was shown in Arabidopsis that several
abiotic and nutritional stresses can co-opt the xylem regulatory network
(Taylor-Teeples et al., 2015). Co-optation of a developmental network is
a mean to facilitate adaptation to stress. If tolerance to stress is important
for all plants because of their sessile lifestyle, it is crucial for long-living trees
which have to cope with seasonal variations and climatic changes all along
their life. Dissecting the mechanisms and the genes underlying stress toler-
ance at the wood level is an important challenge to address in the near future
given the threat of climate changes on forest trees.
Remarkably, in poplar, alternative splicing (through intron retention)
was highlighted as a key mechanism modulating the activities of the
wood-associated NAC-domain TFs with reciprocal cross-regulation of
the VND and SND clades (Li et al., 2012;Lin et al., 2017;Zhao et al.,
2014). Similar splice variants were conserved in Eucalyptus,but none was
found in Arabidopsis. Given the number of alternative splicing events in
the wood transcriptome of both Eucalyptus and Populus (Bao et al., 2013;
Xu et al., 2014) and the fact that abiotic stresses modulate landscape of poplar
transcriptome via alternative splicing, intron retention and isoform ratio
switching (Filichkin et al., 2018), the occurrence of alternative splicing in
regulating the activities wood-associated TFs is one important direction
to explore in the future. Besides more efforts need to be dedicated to dec-
iphering the wood interactome since not all genes expressed in xylem are
regulated at the transcriptional level, and even for those which are regulated
at that level, they are often regulated at the post-transcriptional level (Soler
et al., 2016). The cellular context in which occur interactions with partners
is also an important aspect that needs to be determined.
The large high-resolution profiling of the cambial zone of aspen (Sundell
et al., 2017), which is obviously not possible in Arabidopsis, has allowed to
226 Eduardo L.O. Camargo et al.
build co-expression gene networks or system biology approaches to inves-
tigate, for instance, the control of wood formation by auxin ( Johnsson et al.,
2018) and ethylene (Seyfferth et al., 2018). These powerful tools enabled to
identify “hubs” and modules which point out hormone signalling candi-
dates, and potential target genes and biological processes under the control
of hormones during secondary growth in trees. Using such an approach, it
was possible to establish a central role of the IAA/GA cross-talk in determin-
ing cell fate possibly through the control of SND/NST and VND master
regulators ( Johnsson et al., 2018).
The cross-talk between auxin and other hormones seems to be central at
all stages of secondary xylem differentiation, i.e., from cambium division to
the later stage of SW maturation. This exiting area needs more investigations
to identify the genes and the mechanisms integrating this cross-talk, which is
likely modulated by environmental signals. Technologies enabling fine spa-
tial resolution will be instrumental in the future to get deeper insights in the
regulation of cambial activity and of subsequent xylem differentiation steps.
Finally, new opportunities are created by the development of system
genetic approaches and network modelling (Mizrachi & Myburg, 2016)
which involves multilevel data integration from a population of genetically
diverse individuals and focusses the role of underlying molecular networks in
impacting the relationships between traits. Getting a more comprehensive
view of the molecular networks underlying wood formation will surely
impact tree breeding strategies and genetic engineering approaches to gen-
erate trees more suitable for industrial uses and more adapted to environ-
mental changes.
The authors thank the University Paul Sabatier Toulouse III, the Centre National pour la
Recherche Scientifique and the French Laboratory of Excellence project “TULIP”
(ANR-10-LABX-41; ANR-11-IDEX-0002-02) for their financial support. E.L.O.C.
acknowledges the PDE-CNPq (202228/2015-0) postdoctoral fellowship.
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Further reading
Matsumoto-Kitano, M., Kusumoto, T., Tarkowski, P., Kinoshita-Tsujimura, K.,
Vaclavikova, K., Miyawaki, K., et al. (2008). Cytokinins are central regulators of cambial
activity. Proceedings of the National Academy of Sciences of the United States of America,105,
233Regulation of wood formation
... Subsequent studies have shown that certain NAC domain TFs are master switches controlling SCW formation, including VND1-VND7, NST1-NST3, and SND1 (Zhong et al., 2006;Mitsuda et al., 2007;Yamaguchi et al., 2008), that directly or indirectly regulate the expression of numerous MYB genes. Thus, MYB TFs are the second-layer master regulators of SCW formation, such as AtMYB58/63/85, which are lignin-specific TFs, and AtMYB26/32/41/44/46/61/83/103, which participate in SCW biosynthesis Ye, 2009, 2014;Camargo et al., 2019). ...
... SCW formation is a complex process that requires the coordinated expression of a series of biosynthetic genes (Wang and Dixon, 2012) and thus a complicated transcriptional regulatory network that can activate or inactivate the expression of enzyme genes (Camargo et al., 2019). Over the past decades, a transcriptional regulatory model has been established by primarily using Arabidopsis, in which the NAC and MYB TF families play leading and secondary roles, respectively (Zhong and Ye, 2014). ...
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Wood formation is a complicated process under the control of a large set of transcription factors. NAC transcription factors are considered “master switches” in this process. However, few NAC members have been cloned and characterized in Eucalyptus , which is one of the most economically important woody plants. Here, we reported an NAC transcription factor from Eucalyptus grandis , EgNAC141, which has no Arabidopsis orthologs associated with xylogenesis-related processes. EgNAC141 was predominantly expressed in lignin-rich tissues, such as the stem and xylem. Overexpression of EgNAC141 in Arabidopsis resulted in stronger lignification, larger xylem, and higher lignin content. The expression of lignin biosynthetic genes in transgenic plants was significantly higher compared with wild-type plants. The transient expression of EgNAC141 activated the expression of Arabidopsis lignin biosynthetic genes in a dual-luciferase assay. Overall, these results showed that EgNAC141 is a positive regulator of lignin biosynthesis and may help us understand the regulatory mechanism of wood formation.
... In the last two decades, tremendous progress had been made in our understanding of the transcriptional regulation of the deposition of lignified SCWs Overexpression of MYB58 or its co-ortholog MYB63 specifically activated the monolignol pathway and lignin accumulation at the expense of biomass production (Zhou et al., 2009). Most of the transcriptional regulatory network underlying the lignified SCW is conserved in other plants (Ohtani & Demura, 2019) but differences have been highlighted between Arabidopsis and woody angiosperms for instance (Camargo et al., 2019;Nakano, Yamaguchi, Endo, Rejab, & Ohtani, 2015 and references therein). In addition, the role of lignin-specific SG3-type R2R3-MYBs (MYB58/63) in vascular cell wall lignification seems to be conserved in dicot plants ...
... For each gene, the fourth column provides the closest Arabidopsis thaliana ortholog(s), mentioned in the references cited, or obtained by using BLASTP of NCBI database (best hits >80% of query protein identity).(for reviews seeCamargo, Ployet, Cassan-Wang, Mounet, & Grima- Pettenati, 2019;Grima-Pettenati, Soler, Camargo, & Wang, 2012;Hussey, Mizrachi, Creux, & Myburg, 2013;Schuetz, Smith, & Ellis, 2013;Wang & Dixon, 2012 and reference therein). In Arabidopsis, a complex hierarchical regulatory network has been proposed to control SW deposition in which the Transcription Factors (TF) NAM/ATAF/CUC (NAC) such as NST1, SND1, NST2, VND6 and VND7 act as first-level master switches. ...
The appearance of lignin, 400 million-years ago, has been crucial for the successful land colonization by plants. This complex phenolic polymer which confers hydrophobicity and resistance to degradation to secondary cell walls, is also neosynthesized in response to both biotic and abiotic stresses. Lignin biosynthesis during development is regulated by a complex hierarchical transcriptional gene regulatory network. Notably, distinct abiotic stresses can co-opt different genes of this network, leading to different lignification patterns. Co-optation of this developmental network is a mean to facilitate adaptation to stress by promoting functional adaptation. Supporting this hypothesis, a growing number of transcription factors known to be involved in the regulation of secondary cell wall, were shown to integrate both developmental and environmental signals leading to an increased tolerance to abiotic stresses, such as drought or salinity. On the other hand, some transcription factors known to be involved in stress response were shown to impact lignin biosynthesis as part of the functional adaptation performed by plants to cope with natural constraints. In this review, we focus on the most recent findings on the cross-talk between stress-signaling pathways and regulation of lignin biosynthesis in response to abiotic stress and to a lesser extent to biotic stresses. We consider the transcriptional regulation level which has received much attention and the emerging roles of posttranscriptional and posttranslational regulations in the control of lignin biosynthesis in response to stresses. We end discussing future outlines and challenges to fill gaps in this emerging and exciting research field.
... In secondary growth, i.e., thickening growth, the secondary xylem is produced by the division and differentiation of cambium ( Figure 1). Notably, secondary xylem in the stem, also known as wood, is the main source of tree biomass, which can be used for multiple purposes, such as papermaking, furniture, construction and biofuels [4]. In addition, carbon storage in wood is also crucial for balancing the level of carbon dioxide in the atmosphere [5]. ...
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Xylem, as a unique organizational structure of vascular plants, bears water transport and supports functions necessary for plant survival. Notably, secondary xylem in the stem (i.e., wood) also has important economic and ecological value. In view of this, the regulation of xylem development has been widely concerned. In recent years, studies on model plants Arabidopsis and poplar have shown that transcription factors play important regulatory roles in various processes of xylem development, including the directional differentiation of procambium and cambium into xylem, xylem arrangement patterns, secondary cell wall formation and programmed cell death. This review focuses on the regulatory roles of widely and thoroughly studied HD-ZIP, MYB and NAC transcription factor gene families in xylem development, and it also pays attention to the regulation of their upstream microRNAs. In addition, the existing questions in the research and future research directions are prospected.
... However, unlike the herbaceous annual Arabidopsis, perennial woody plants exhibit extreme secondary growth, characterized by formation of secondary cell walls (SCW) and additional dynamic seasonal changes influenced by various environmental stresses [11,12]. Thus, partly due to reliance on information and markers from Arabidopsis, the molecular-level regulation of vascular tissue development in trees, especially the SCW regulatory network involved, has not been fully elucidated [10,13]. ...
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Background Plant secondary growth depends on the activity of the vascular cambium, which produces xylem and phloem. Wood derived from xylem is the most abundant form of biomass globally and has played key socio-economic and subsistence roles throughout human history. However, despite intensive study of vascular development, the full diversity of cell types and the gene networks engaged are still poorly understood. Results Here, we have applied an optimized protoplast isolation protocol and RNA sequencing to characterize the high-resolution single-cell transcriptional landscape of highly lignified poplar stems. We identify 20 putative cell clusters with a series of novel cluster-specific marker genes and find that these cells are highly heterogeneous based on the transcriptome. Analysis of these marker genes’ expression dynamics enables reconstruction of the cell differentiation trajectories involved in phloem and xylem development. We find that different cell clusters exhibit distinct patterns of phytohormone responses and emphasize the use of our data to predict potential gene redundancy and identify candidate genes related to vascular development in trees. Conclusions These findings establish the transcriptional landscape of major cell types of poplar stems at single-cell resolution and provide a valuable resource for investigating basic principles of vascular cell specification and differentiation in trees.
... Cette représentation schématique se base sur une revue extensive de (Camargo et al., 2019). Plusieurs facteurs peuvent influencer l'expression de ces gènes. ...
Les arbres atteignent des hauteurs et des durées de vie considérables grâce aux propriétés remarquables de leur bois. En effet, le bois remplit trois fonctions principales : (i) la conduction de la sève brute de la racine au houppier, (ii) le soutien mécanique de la masse toujours en augmentation de l'arbre en croissance et (iii) le stockage de réserves temporaires, capitales pour la pérennité de l'arbre. Chez les angiospermes, les vaisseaux, les fibres et les rayons parenchymateux sont, respectivement, affiliés à ces fonctions. Chacune de ces cellules possède son propre schéma de développement. Par ailleurs, la composition et la structure des parois de ces cellules varient considérablement en fonction des stades de développement et des conditions environnementales. Cette complexité représente donc un frein à l’étude des mécanismes moléculaires de la formation du bois. Cette difficulté peut être contournée par le développement d’approches à l’échelle cellulaire. La thèse présentée ici vise à une caractérisation du développement des fibres, plus particulièrement de leurs parois secondaires, par le déploiement d’outils de caractérisation à l’échelle cellulaire et d’une analyse intégrative à cette échelle. Le développement d’une méthode de caractérisation des parois à l’échelle cellulaire, l’imagerie hyperspectrale en ATR-FTIR, a permis une analyse fine des différences entre types cellulaires au sein d’un arbre et entre types de bois pour un même type cellulaire. L’étude de données transcriptomiques obtenues par RNA-Seq de fibres et rayons microdisséqués a, elle, permis d’identifier des différences transcriptionnelles entre ces deux types cellulaires. La combinaison de ces deux résultats a permis d’identifier des acteurs semblant majeurs dans le développement des fibres de bois. Ce travail de thèse ouvre donc des perspectives de recherche permettant de mieux comprendre les mécanismes moléculaires associés à la formation des fibres de bois. Title: Towards the development of a model of secondary cell wall formation of wood fibre in poplar Trees are able to grow high et survive many years thanks to their wood properties. Wood delivers three major functions in trees: (i) water conduction, (ii) mechanical support et (iii) nutrient storage. In Angiosperm trees, vessels, fibers et parenchyma rays are respectively assigned to these functions, each of them following their own development scheme. Cell wall composition et structure varies greatly depending on cell type, developmental stage et environmental conditions. This complexity therefore represents a hindrance to study the molecular mechanisms of wood formation. However, this can be circumvented by the development of cell-specific approaches. This work aims at characterizing fiber development, focusing on their secondary cell wall, developing cell-specific methods et integrative analysis at the cell level. Development of ATR-FTIR hyperspectral imaging enabled to finely characterize differences in cell wall composition between cell types in a tree et within cell types in different types of wood. Transcriptomics data obtained by RNA-Seq of microdissected fibers et rays gave rise to major differences in the transcriptome of these two cell types. Combining both kind of result led to the identification of key players in fibers development. Hence, this work opens up new research hypothesis, which could lead to a better understanding of the molecular mechanisms underlying wood fiber development, including from a dynamic perspective.
... PtrSND1-A2 IR is derived from PtrSND1-A2 (also named PtrWND1B/PtrVNS11), having lost its DNA binding and activation domain but retaining its dimerization capability; it represses the transcription of PtrSND1 members and their target gene PtrMYB021 by translocating into the nucleus exclusively as a heterodimeric partner with full-size PtrSND1s (Li et al., 2012). This is the first time to report about the TF family's auto-repression by its own splice variant found in plants (Zhang et al., 2018;Camargo et al., 2019). Other research has shown that overexpression of PtrSND1-A2 enhances fiber cell wall thickening, while overexpression of PtrSND1-A2 IR (PtrWND1Bl) inhibits the fiber cell wall-thickening process (Zhao et al., 2014). ...
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Enormous distinctions of the stem structure and cell types between gymnosperms and angiosperms tree species are expected to cause quite different wood physical and mechanical attributes, however, the molecular mechanisms underlying the differing wood morphology are still unclear. In this study, we compared the transcriptomes obtained by RNA-Seq between Populus alba × P. glandulosa clone 84K, and Larix kaempferi (Lamb.) Carr trees. Available genome resource served as reference for P. alba × P. glandulosa and the Iso-Seq results of a three-tissues mixture (xylem, phloem, and leaf) were used as the reference for L. kaempferi to compare the xylem-specifically expressed genes and their alternative splicing model. Through screening, we obtained 13,907 xylem-specifically expressed genes (5,954 up-regulated, 7,953 down-regulated) in the xylem of P. alba × P. glandulosa, and 2,596 xylem-specifically expressed genes (1,648 up-regulated, 948 down-regulated) in the xylem of L. kaempferi. From the GO and KEGG analyses, some genes associated with two wood formation-related pathways, namely those for phenylpropanoid biosynthesis, and starch and sucrose metabolism, were successfully screened. Then the distributions and gene expression models between P. alba × P. glandulosa and L. kaempferi in those pathways were compared, which suggested differential wood formation processes between the angiosperm and gymnosperm trees. Furthermore, a Weight Gene Co-expression Network Analysis (WGCNA) for total xylem-specifically expressed genes in two species was conducted, from which wood formation-related modules were selected to build a co-expression network for the two tree species. The genes within this co-expression network showed different co-expression relationships between the angiosperm and gymnosperm woody species. Comparing the alternative splicing events for wood formation-related genes suggests a different post-transcriptional regulation process exists between the angiosperm and gymnosperm trees. Our research thus provides the foundation for the in-depth investigation of different wood formation mechanisms of angiosperm and gymnosperm species.
... The effects of abiotic stresses on SCW biogenesis and wood formation have been covered in other reviews (Moura et al., 2010;Houston et al., 2016;Camargo et al., 2019;Eckert et al., 2019). Although few studies have investigated the role of SCW synthesis genes in stress responses in trees, many transcriptomic studies suggest roles for SCW biosynthesis in both seasonal adaptations and abiotic stress responses, with consequential effects on biomass utilization (Fox et al., 2017;Ployet et al., 2017;Wildhagen et al., 2017;Jokipii-Lukkari et al., 2018). ...
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A major challenge for sustainable food, fuel, and fiber production is simultaneous genetic improvement of yield, biomass quality, and resilience to episodic environmental stress and climate change. For Populus and other forest trees, quality traits involve alterations in the secondary cell wall (SCW) of wood for traditional uses, as well as for a growing diversity of biofuels and bioproducts. Alterations in wood properties that are desirable for specific end uses can have negative effects on growth and stress tolerance. Understanding of the diverse roles of SCW genes is necessary for the genetic improvement of fast-growing, short-rotation trees that face perennial challenges in their growth and development. Here, we review recent progress into the synergies and antagonisms of SCW development and abiotic stress responses, particularly, the roles of transcription factors, SCW biogenesis genes, and paralog evolution.
... Plant hormones have been identified as key regulators in longitudinal and radial growth by influencing cambial acitivity and tissue differentiation process in higher plants (Du et al. 2020). Among these, auxins, gibberellins and cytokinins are reported to play an active role in cell division, elongation and differentiation process (Sharma and Zheng 2019;Camargo et al. 2018;Butto et al. 2020). These Handling Editor: Pramod Kumar Nagar. ...
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Plant growth regulators play a key role in cell wall structure and chemistry of woody plants. Understanding of these regulatory signals is important in advanced research on wood quality improvement in trees. The present study is aimed to investigate the influence of exogenous application of 24-epibrassinolide (EBR) and brassinosteroid inhibitor, brassinazole (BRZ) on wood formation and spatial distribution of cell wall polymers in the xylem tissue of Leucaena leucocephala using light and immuno electron microscopy methods. Brassinazole caused a decrease in cambial activity, xylem differentiation, length and width of fibres, vessel element width and radial extent of xylem suggesting brassinosteroid inhibition has a concomitant impact on cell elongation, expansion and secondary wall deposition. Histochemical studies of 24-epibrassinolide treated plants showed an increase in syringyl lignin content in the xylem cell walls. Fluorescence microscopy and transmission electron microscopy studies revealed the inhomogenous pattern of lignin distribution in the cell corners and middle lamellae region of BRZ treated plants. Immunolocalization studies using LM10 and LM 11 antibodies have shown a drastic change in the micro-distribution pattern of less substituted and highly substituted xylans in the xylem fibres of plants treated with EBR and BRZ. In conclusion, present study demonstrates an important role of brassinosteroid in plant development through regulating xylogenesis and cell wall chemistry in higher plants.
... The hormone auxin is involved in regulating virtually all morphogenetic processes in plant organisms, and its role in conducting tissues differentiation in tree trunks is well known [1][2][3][4][5][6][7][8]. Auxin contribution to the regulation of morphogenetic processes largely depends on its concentration and distribution in plant organs and tissues. ...
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Auxin status in woody plants is believed to be a critical factor for the quantity and quality of the wood formed. It has been previously demonstrated that figured wood formation in Karelian birch (Betula pendula Roth var. carelica (Merckl.) Hämet-Ahti) is associated with a reduced auxin level and elevated sugar content in the differentiating xylem, but the molecular mechanisms of the abnormal xylogenesis remained largely unclear. We have identified genes involved in auxin biosynthesis (Yucca), polar auxin transport (PIN) and the conjugation of auxin with amino acids (GH3) and UDP-glucose (UGT84B1) in the B. pendula genome, and analysed their expression in trunk tissues of trees differing in wood structure. Almost all the investigated genes were overexpressed in Karelian birch trunks. Although Yucca genes were overexpressed, trunk tissues in areas developing figured grain had traits of an auxin-deficient phenotype. Overexpression of GH3s and UGT84B1 appears to have a greater effect on figured wood formation. Analysis of promoters of the differentially expressed genes revealed a large number of binding sites with various transcription factors associated with auxin and sugar signalling. These data agree with the hypothesis that anomalous figured wood formation in Karelian birch may be associated with the sugar induction of auxin conjugation.
Secondary xylem is the chief form of lignocellulosic biomass on the planet and consists mainly of fibres, vessels and tracheids with lignified secondary cell walls (SCWs). These cells develop from xylem mother cells produced from the meristematic vascular cambium, undergoing cell expansion, SCW deposition, programmed cell death and lignification within a narrow developmental zone. Understanding the transcriptional control of this complex process has been an activate area of research for over two decades, where significant contributions in the model annual Arabidopsis thaliana (Arabidopsis) and the model tree genus Populus have provided detailed insights into a semi-hierarchical transcriptional regulatory network (TRN) underlying SCW deposition and lignification. In this chapter, I summarise the classical three-tiered SCW TRN model and elaborate on its expanded structure, discussing key transcription factors underlying lignin formation in Arabidopsis. I present a comprehensive new model of the SCW TRN of poplar, with a commentary on the conservation and divergence of the SCW TRN across plant lineages and the possible existence of lineage-specific regulators that act as “accessory” components to the core TRN. Finally, I discuss key post-translational modifications that affect the activity of the transcription factors comprising the SCW TRN and recent insights into cooperation and antagonism between transcription factors through the formation of protein complexes. We are learning that the SCW TRN has undergone substantial re-wiring in well-studied models and that plants have adopted a myriad of combinatorial and post-translational mechanisms to modulate SCW transcriptional control according to particular biological contexts.
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We used sector analysis to study cambium development and dynamics and to test if fundamental developmental and functional differences exist between cambial initials as true ‘stem cells’ and more differentiated mother cells. In many higher plants, a cylindrical lateral meristem, the vascular cambium, forms along the plant axis. Most notably in stems of perennial tree species, this meristem gives rise to xylem (wood) towards the inside of the trunk and phloem (bark) towards the outside. As such, the vascular cambium is responsible for the production of most of the planet’s forest biomass, significantly contributing to the global carbon cycle. Using the bacterial uid A reporter gene in Agrobacterium-based in vivo stem transformation experiments in poplar trees we created 379 cambium sectors that originated from the transformation of individual cells. Results from our analysis of sector frequency and patterns are consistent with the poplar cambium featuring a single layer of true cambial initials (being able to divide both anti- and periclinally). We show that initials are frequently lost from the cambium, that such cell loss rarely occurs at mother cell level, that phloem and xylem differentiation are controlled independently, and that the frequency of mother cell replenishment is not predetermined.
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Wood formation in higher plants is a complex and costly developmental process regulated by a complex network of transcription factors, short peptide signals and hormones. Correct spatiotemporal initiation of differentiation and downstream developmental stages is vital for proper wood formation. Members of the NAC (NAM, ATAF1/2, and CUC) family of transcription factors are described as top level regulators of xylem cell fate and secondary cell wall (SCW) deposition, but the signals initiating their transcription have yet to be elucidated. We found that treatment of Populus stems with auxin repressed transcription of NAC transcription factors associated with fibre and SCW formation, and induced vessel‐specific NACs, whereas gibberellic acid induced the expression of both classes of NAC domain transcription factors involved in wood formation. These transcriptional changes were reflected in alterations of stem anatomy, i.e. auxin treatment reduced cell wall thickness, whereas gibberellic acid had a promotive effect on SCW deposition and the rate of wood formation. Similar changes were observed on treatment of Arabidopsis thaliana stems with gibberellic acid (GA) or the synthetic auxin NAA. We also observed corresponding changes in PIN5 over‐expressing lines, where interference with auxin transport leads to premature SCW deposition and formation of additional fibre bundles. Together, this suggests wood formation is regulated by an integrated readout of both auxin and gibberellic acid which, in turn controls expression of fibre and vessel specific NACs. This article is protected by copyright. All rights reserved.
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Premise of the study: Dimensions and spatial distribution of vessels are critically important features of woody stems, allowing for adaptation to different environments through their effects on hydraulic efficiency and vulnerability to embolism. Although our understanding of vessel development is poor, basipetal transport of auxin through the cambial zone may play an important role. Methods: Stems of Populus tremula ×alba were treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) in a longitudinal strip along the length of the lower stem. Vessel lumen diameter, circularity, and length; xylem growth; tension wood area; and hydraulic conductivity before and after a high pressure flush were determined on both NPA-treated and control plants. Key results: NPA-treated stems formed aberrant vessels that were short, small in diameter, highly clustered, and angular in cross section, whereas xylem formed on the untreated side of the stem contained typical vessels that were similar to those of controls. NPA-treated stems had reduced specific conductivity relative to controls, but this difference was eliminated by the high-pressure flush. The control treatment (lanolin + dimethyl sulfoxide) reduced xylem growth and increased tension wood formation, but never produced the aberrant vessel patterning seen in NPA-treated stems. Conclusions: These results are consistent with a model of vessel development in which basipetal polar auxin transport through the xylem-side cambial derivatives is required for proper expansion and patterning of vessels and demonstrate that reduced auxin transport can produce stems with altered stem hydraulic properties.
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Spatial organization of signalling events of the phytohormone auxin is fundamental for maintaining a dynamic transition from plant stem cells to differentiated descendants. The cambium, the stem cell niche mediating wood formation, fundamentally depends on auxin signalling but its exact role and spatial organization is obscure. Here we show that, while auxin signalling levels increase in differentiating cambium descendants, a moderate level of signalling in cambial stem cells is essential for cambium activity. We identify the auxin-dependent transcription factor ARF5/MONOPTEROS to cell-autonomously restrict the number of stem cells by directly attenuating the activity of the stem cell-promoting WOX4 gene. In contrast, ARF3 and ARF4 function as cambium activators in a redundant fashion from outside of WOX4-expressing cells. Our results reveal an influence of auxin signalling on distinct cambium features by specific signalling components and allow the conceptual integration of plant stem cell systems with distinct anatomies.
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Thickening of tree stems is the result of secondary growth, accomplished by the meristematic activity of the vascular cambium. Secondary growth of the stem entails developmental cascades resulting in the formation of secondary phloem outwards and secondary xylem (i.e. wood) inwards of the stem. Signaling and transcriptional reprogramming by the phytohormone ethylene modifies cambial growth and cell differentiation, but the molecular link between ethylene and secondary growth remains unknown. We addressed this shortcoming by analyzing expression profiles and co-expression networks of ethylene pathway genes using the AspWood transcriptome database which covers all stages of secondary growth in aspen (Populus tremula) stems. ACC synthase expression suggests that the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is synthesized during xylem expansion and xylem cell maturation. Ethylene-mediated transcriptional reprogramming occurs during all stages of secondary growth, as deduced from AspWood expression profiles of ethylene-responsive genes. A network centrality analysis of the AspWood dataset identified EIN3D and eleven ERFs as hubs. No overlap was found between the co-expressed genes of the EIN3 and ERF hubs, suggesting target diversification and hence independent roles for these transcription factor families during normal wood formation. The EIN3D hub was part of a large co-expression gene module, which contained 16 transcription factors, among them several new candidates that have not been earlier connected to wood formation and a VND-INTERACTING 2 (VNI2) homolog. We experimentally demonstrated EIN3D function in ethylene signaling in Arabidopsis thaliana. The ERF hubs ERF118 and ERF119 were connected on the basis of their expression pattern and gene co-expression module composition to xylem cell expansion and secondary cell wall formation, respectively. We hereby establish data resources for ethylene-responsive genes and potential targets for EIN3D and ERF transcription factors in Populus stem tissues, which can help to understand the range of ethylene targeted biological processes during secondary growth
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Abiotic stresses affect plant physiology, development, growth, and alter pre-mRNA splicing. Western poplar is a model woody tree and a potential bioenergy feedstock. To investigate the extent of stress-regulated alternative splicing (AS), we conducted an in-depth survey of leaf, root, and stem xylem transcriptomes under drought, salt, or temperature stress. Analysis of approximately one billion of genome-aligned RNA-Seq reads from tissue- or stress-specific libraries revealed over fifteen millions of novel splice junctions. Transcript models supported by both RNA-Seq and single molecule isoform sequencing (Iso-Seq) data revealed a broad array of novel stress- and/or tissue-specific isoforms. Analysis of Iso-Seq data also resulted in the discovery of 15,087 novel transcribed regions of which 164 show AS. Our findings demonstrate that abiotic stresses profoundly perturb transcript isoform profiles and trigger widespread intron retention (IR) events. Stress treatments often increased or decreased retention of specific introns – a phenomenon described here as differential intron retention (DIR). Many differentially retained introns were regulated in a stress- and/or tissue-specific manner. A subset of transcripts harboring super stress-responsive DIR events showed persisting fluctuations in the degree of IR across all treatments and tissue types. To investigate coordinated dynamics of intron-containing transcripts in the study we quantified absolute copy number of isoforms of two conserved transcription factors (TFs) using Droplet Digital PCR. This case study suggests that stress treatments can be associated with coordinated switches in relative ratios between fully spliced and intron-retaining isoforms and may play a role in adjusting transcriptome to abiotic stresses.
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Secondary cell wall (SCW) biosynthesis is the biological process that generates wood, an important renewable feedstock for materials and energy. NAC domain transcription factors, particularly Vascular-Related NAC-Domain (VND) and Secondary Wall-Associated NAC Domain (SND) proteins, are known to regulate SCW differentiation. The regulation of VND and SND is important to maintain homeostasis for plants to avoid abnormal growth and development. We previously identified a splice variant, PtrSND1-A2 IR , derived from PtrSND1-A2 as a dominant-negative regulator, which suppresses the transactivation of all PtrSND1 family members. PtrSND1-A2IR also suppresses the self-activation of the PtrSND1 family members except for its cognate transcription factor, PtrSND1-A2, suggesting the existence of an unknown factor needed to regulate PtrSND1-A2 Here, a splice variant, PtrVND6-C1 IR , derived from PtrVND6-C1 was discovered that suppresses the protein functions of all PtrVND6 family members. PtrVND6-C1IR also suppresses the expression of all PtrSND1 members, including PtrSND1-A2, demonstrating that PtrVND6-C1IR is the previously unidentified regulator of PtrSND1-A2 We also found that PtrVND6-C1IR cannot suppress the expression of its cognate transcription factor, PtrVND6-C1PtrVND6-C1 is suppressed by PtrSND1-A2IR Both PtrVND6-C1IR and PtrSND1-A2IR cannot suppress their cognate transcription factors but can suppress all members of the other family. The results indicate that the splice variants from the PtrVND6 and PtrSND1 family may exert reciprocal cross-regulation for complete transcriptional regulation of these two families in wood formation. This reciprocal cross-regulation between families suggests a general mechanism among NAC domain proteins and likely other transcription factors, where intron-retained splice variants provide an additional level of regulation.
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Cellular processes, such as signal transduction and cell wall deposition, are organized by macromolecule interactions. Experimentally determined protein-protein interactions (PPIs) and protein-DNA interactions (PDIs) relevant to woody plant development are sparse. To begin to develop a Populus trichocarpa Torr. & A. Gray wood interactome, we applied the yeast-two-hybrid (Y2H) assay in different ways to enable the discovery of novel PPIs and connected networks. We first cloned open reading frames (ORFs) for 361 genes markedly upregulated in secondary xylem compared with secondary phloem and performed a binary Y2H screen with these proteins. By screening a xylem cDNA library for interactors of a subset of these proteins and then recapitulating the process by using a subset of the interactors as baits, we ultimately identified 165 PPIs involving 162 different ORFs. Thirty-eight transcription factors (TFs) included in our collection of P. trichocarpa wood ORFs were used in a Y1H screen for binding to promoter regions of three genes involved in lignin biosynthesis resulting in 40 PDIs involving 20 different TFs. The network incorporating both the PPIs and PDIs included 14 connected subnetworks, with the largest having 132 members. Protein-protein interactions and PDIs validated previous reports and also identified new candidate wood formation proteins and modules through their interactions with proteins and promoters known to be involved in secondary cell wall synthesis. Selected examples are discussed including a PPI between Mps one binder (MOB1) and a mitogen-activated protein kinase kinase kinase kinase (M4K) that was further characterized by assays confirming the PPI as well as its effect on subcellular localization. Mapping of published transcriptomic data showing developmentally detailed expression patterns across a secondary stem onto the network supported that the PPIs and PDIs are relevant to wood formation, and also illustrated that wood-associated interactions involve gene products that are not upregulated in secondary xylem.
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Wood formation is a complex developmental process under multi-level transcriptional control executed by a large set of transcription factors. However, only limited members have been characterized to be key regulators of lignin biosynthesis in poplar. Here we report the conserved and unique functions of PtoMYB170, a transcription factor identified from Populus tomentosa (Chinese white poplar), in lignin deposition and drought tolerance in comparison with its duplicate paralog PtoMYB216. PtoMYB170 is preferentially expressed in young leaves and xylem tissues. Overexpression of PtoMYB170 in transgenic poplar plants resulted in stronger lignification and more thickened secondary wall in xylem compared with wild-type plants, whereas the CRISPR/Cas9-generated mutation of PtoMYB170 weakened lignin deposition, thereby leading to a more flexible and collapsed xylem phenotype. Transient expression experiments demonstrated that PtoMYB170 specifically activated the expression of lignin biosynthetic genes, consistent with the function of PtoMYB216. However, GUS staining assays revealed that PtoMYB170 was specifically expressed in guard cells of transgenic Arabidopsis while PtoMYB216 was not. Heterologous expression of PtoMYB170 in Arabidopsis enhanced stomatal closure in the dark and resulted in drought tolerance of the transgenic plants through reduced water loss, indicating a diversified role from PtoMYB216. These results revealed the PtoMYB170-dependent positive transcriptional regulation on lignin deposition in poplar and its coordinated function in enhancing drought tolerance by promoting dark-induced stomatal closure.
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Trees represent the largest terrestrial carbon sink and a renewable source of ligno-cellulose. There is significant scope for yield and quality improvement in these largely undomesticated species, and efforts to engineer elite varieties will benefit from improved understanding of the transcriptional network underlying cambial growth and wood formation. We generated high-spatial-resolution RNA sequencing data spanning the secondary phloem, vascular cambium and wood forming tissues of Populus tremula. The transcriptome comprised 28,294 expressed, annotated genes, 78 novel protein-coding genes and 567 putative long intergenic non-coding RNAs. Most paralogs originating from the Salicaceae whole genome duplication had diverged expression, with the exception of those highly expressed during secondary cell wall deposition. Co-expression network analyses revealed that regulation of the transcriptome underlying cambial growth and wood formation comprises numerous modules forming a continuum of active processes across the tissues. A comparative analysis revealed that a majority of these modules are conserved in Picea abies. The high spatial resolution of our data enabled identification of novel roles for characterized genes involved in xylan and cellulose biosynthesis, regulators of xylem vessel and fiber differentiation and lignification. An associated web resource (AspWood, provides interactive tools for exploring the expression profiles and co-expression network.