Digging in wood: New insights
in the regulation of wood
formation in tree species
Eduardo L.O. Camargo
, Raphaël Ployet
, Hua Cassan-Wang
, Jacqueline Grima-Pettenati
Laboratoire de Recherche en Sciences Vegetales, Universite de Toulouse, CNRS, UPS,
´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: firstname.lastname@example.org
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 Protein–protein interactions 224
5. Concluding remarks 225
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 trees’wood-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.
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
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
peptide CLE41 (CLAVATA3/EMBRYO SURROUNDING REGION-
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
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
showed the AUXIN RESPONSE FACTORs ARF3,ARF4 and
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
2.3 The cross-talk between auxin and cytokinins stimulates
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 (Bj€orklund, 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 (Bj€orklund 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
Saidi, Hefer, Myburg, &
Grima-Pettenati, 2015) Putative poplar co-orthologs References
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/
NST2 (ANAC066) — —
VND VND1 (ANAC037)
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) — —
Ohtani et al. (2011),Zhong et al.
(2010a, 2010b),Li et al. (2012),
Johnsson et al. (2018)
VND6 (ANAC101) EgrNAC26 –PtVNS05/PtrWND3A/PtrVND6-
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)
Grant, Fujino, Beers, and Brunner
(2010),Johnsson et al. (2018)
SND3 (ANAC010) EgrNAC44
Grant et al. (2010),Johnsson et al.
XND1 (ANAC104) EgrNAC137
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://
aspwood.popgenie.org/), 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
INSENSITIVE 3D)and11ERFs (ETHYLENE RESPONSE FACTORS)
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
ReferencesLignin Cellulose Xylan
BplMYB46 KP711284 AtMYB46 BplPAL, Bpl4CL,
Guo et al.
EgMYB1 CAE09058 AtMYB4 EgCCR, EgCAD2, PtrPAL2,
Legay et al.
EgMYB2 AJ576023 AtMYB46 NtPAL, NtC4H, Nt4CL,
NtC3H, NtHCT, NtCOMT,
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,
et al. (2010),
Zhong et al.
PtrMYB3 Potri.001G267300 AtMYB46/83 PtrCOMT1,
et al. (2010),
Zhong et al.
Table 2 MYB transcription factors from angiosperm trees involved in SW formation.—cont’d
species TFs Gene ID
ReferencesLignin Cellulose Xylan
PtrMYB20 Potri.009G061500 AtMYB46/83 PtrCOMT1,
et al. (2010),
Zhong et al.
PtrMYB21 Potri.009G053900 AtMYB46/83 PtrCOMT1,
et al. (2010),
Zhong et al.
PtrMYB152 Potri.017G130300 AtMYB43/20 At4CL1, AtHCT, AtC3H1,
PtrPAL4, Pt4CL3, Ptr4CL5,
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,
Xu et al.
PtoMYB216 JQ801749 AtMYB61 PtoPAL4, Pto4CL5,
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
family derived from PtrVND6-C1 (PtVNS01/
PtrWND5A). It suppresses the protein functions of all PtrVND6 family
223Regulation of wood formation
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
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 Protein–protein interactions
Despite the importance of combinatorial control in transcriptional regulation
in plants, there are only a handful of examples of the involvement of
protein–protein 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
protein–protein 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-
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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