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Auxin-mediated Aux/IAA-ARF-HB signaling cascade regulates
secondary xylem development in Populus
Changzheng Xu
1
*, Yun Shen
1
*,FuHe
1
*, Xiaokang Fu
1
*, Hong Yu
1,2
*, Wanxiang Lu
1
, Yongli Li
1
, Chaofeng Li
1,3
,
Di Fan
1
, Hua Cassan Wang
4
and Keming Luo
1
1
Chongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, School of Life Sciences, Southwest University, Chongqing 400715, China;
2
School of Basic Medical
Sciences, Southwest Medical University, Luzhou, Sichuan 646000, China;
3
Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese
Academy of Sciences, Xining 810008, China;
4
UMR5546, Laboratoire de Recherche en Sciences Vegetales, Universite de Toulouse III Paul Sabatier, CNRS, UPS, 31326, Castanet-Tolosan,
France
Authors for correspondence:
Hua Cassan Wang
Tel: +33 5 34323851
Email: huawang76@yahoo.com
Keming Luo
Tel: +86 23 68253021
Email: kemingl@swu.edu.cn
Received: 13 November 2018
Accepted: 14 December 2018
New Phytologist (2019)
doi: 10.1111/nph.15658
Key words: ARF5, Aux/IAA9, auxin, HD-ZIP
III transcription factors, Populus, xylem
development.
Summary
Wood development is strictly regulated by various phytohormones and auxin plays a central
regulatory role in this process. However, how the auxin signaling is transducted in developing
secondary xylem during wood formation in tree species remains unclear.
Here, we identified an Aux/INDOLE-3-ACETIC ACID 9 (IAA9)-AUXIN RESPONSE FACTOR
5 (ARF5) module in Populus tomentosa as a key mediator of auxin signaling to control early
developing xylem development.
PtoIAA9, a canonical Aux/IAA gene, is predominantly expressed in vascular cambium and
developing secondary xylem and induced by exogenous auxin. Overexpression of PtoIAA9m
encoding a stabilized IAA9 protein significantly represses secondary xylem development in
transgenic poplar. We further showed that PtoIAA9 interacts with PtoARF5 homologs via the
C-terminal III/IV domains. The truncated PtoARF5.1 protein without the III/IV domains
rescued defective phenotypes caused by PtoIAA9m. Expression analysis showed that the
PtoIAA9-PtoARF5 module regulated the expression of genes associated with secondary
vascular development in PtoIAA9m- and PtoARF5.1-overexpressing plants. Furthermore,
PtoARF5.1 could bind to the promoters of two Class III homeodomain-leucine zipper (HD-ZIP
III) genes, PtoHB7 and PtoHB8, to modulate secondary xylem formation.
Taken together, our results suggest that the Aux/IAA9-ARF5 module is required for auxin
signaling to regulate wood formation via orchestrating the expression of HD-ZIP III transcrip-
tion factors in poplar.
Introduction
Meristems, specialized structures of reservoirs of stem cells under-
going proliferation, drive postembryonic developmental pro-
grams in plants (Nakajima & Benfey, 2002). In comparison with
primary/longitudinal growth of shoots and roots mediated by
apex-localized meristems, secondary/radial growth is dominated
by vascular cambium (Campbell & Turner, 2017). Cambial cells
sequentially undergo proliferation and differentiation into sec-
ondary vascular tissues (Matte Risopatron et al., 2010). Different
from herbaceous plants, trees exhibit perennial stem thickening
during their life cycle due to continuous competence of the vas-
cular cambium (Sanchez et al., 2012). The secondary xylem,
commonly called wood, overwhelmingly contributes to stem
thickening of trees (Sanchez et al., 2012). Wood production is a
predominant proportion of biomass accumulation in terrestrial
ecosystems and is also of outstanding economic value (Ragauskas
et al., 2006; Bonan, 2008).
Cambium-originated wood formation starts with specification
of secondary xylem cell precursors, which in turn undergo a series
of cellular events for maturation, including expansion, secondary
cell-wall deposition and programmed cell death (Ye & Zhong,
2015). Cambial homeostasis between stem cell identity and
xylem specification affects the deposition of xylem cell layers
(Campbell & Turner, 2017). Expansion of newly specified xylem
cells contributes to the size of woody elements, while secondary
wall deposition is important for functional maturation of xylem.
Although vessels and fibers are lignified components of secondary
xylem, they differ in cell size, secondary cell-wall deposition and
cell viability (Fukuda, 2004). These differences result from the
cellular changes in cytoskeletal arrangement, secondary wall pat-
terning and autolysis that occur during differentiation of trac-
heary elements, the unit of xylem vessels (Fukuda, 2004; Turner
et al., 2007). These coordinated behaviors of cambial cells and
*These authors contributed equally to this work.
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their differentiating descendants are intricately and dynamically
orchestrated by various genetic and environmental factors
(Dejardin et al., 2010; Ye & Zhong, 2015). Recent high-spatial-
resolution profiling analyses in Populus have revealed three tran-
scriptome reprogramming events coinciding with transitions of
distinct cellular behaviors in the context of wood formation (Sun-
dell et al., 2017). Although a comprehensive picture of the gene
regulatory machinery involved in secondary growth has emerged
in recent decades, precise regulation of spatially organized and
temporally coordinated cellular events for wood differentiation
remains largely unknown.
Auxin is a crucial phytohormone for cell–cell communication in
meristems (Leyser, 2005). Auxin signaling is able to direct cellular
behaviors, including cell division, expansion and differentiation, for
various developmental programs (Vanneste & Friml, 2009; Perrot-
Rechenmann, 2010). Previous studies have shown that auxin accu-
mulates in a radial gradient pattern across wood-forming tissues
with a peak in concentration in cambium and bilateral decay
towards differentiating secondary xylem and phloem (Tuominen
et al., 1997; Uggla et al., 1998; Immanen et al., 2016). Auxin is
thus considered as a primary regulator of secondary xylem forma-
tion in a morphogen-like manner (Uggla et al., 1996; Sundberg
et al., 2000). In Pinus, a lack of auxin supply from shoot apex leads
to a loss of fusiform shape of cambial derivatives (Savidge, 1983).
Introduction of a stabilized version of PttIAA3 (homolog of
IAA20s in Arabidopsis and Populus; Supporting Information
Fig. S1) that represses auxin response in hybrid aspen attenuates
periclinal division of cambial cells, but enhances stem cell character-
istics as indicated by an enlarged zone of anticlinal cell division in
cambium (Nilsson et al.,2008).Thesefindingsrevealadualroleof
auxin in coordinating cambial identity and proliferation activity.
Recently, detection of auxin signaling via a high-affinity sensor
indicates its moderate level in cambial stem cells, while increased
level in differentiating cambial descendants (Brackmann et al.,
2018). These results implicate the key role of auxin signaling in
cambial differentiation. Consistently, exogenous auxin is able to
induce differentiation of intact and functionally normal secondary
xylem cells (Bj€orklund et al., 2007). Local enhancement of auxin
signaling is required for wood formation, also supporting the action
of auxin in recruiting cells for differentiation (Bargmann et al.,
2013; Muller et al., 2016). However, the precise regulation of auxin
signaling on these coordinated cellular events for wood differentia-
tion remains obscure in trees.
Auxin perception starts with auxin binding to TIR1
(TRANSPORT INHIBITOR RESPONSE1)/AFB (AUXIN
SIGNALING F-BOX) receptors, and leads to subsequent degra-
dation of the Aux/IAA proteins that repress auxin signaling via
physical interactions with auxin response factor (ARF) proteins
(Mockaitis & Estelle, 2008). The auxin-stimulated protein
turnover of Aux/IAAs releases the transcriptional activity of their
partner ARFs to activate downstream auxin responsive gene
expression (Vanneste & Friml, 2009). Different Aux/IAA-
ARF modules are known to regulate corresponding auxin-
responsive genes and developmental processes (Vanneste &
Friml, 2009). In Arabidopsis, the IAA12/BODENLOS
(BDL)-ARF5/MONOPTEROS (MP) module was identified to
control provascular specification and patterning during embryo-
genesis (Hardtke & Berleth, 1998; Hamann et al., 2002). ARF5/
MP is also crucial for leaf (pre)procambial specification during
leaf vein patterning (Przemeck et al., 1996). In hybrid aspen,
eight Aux/IAA members were assayed for expression of auxin
responsive genes in wood tissues and during transitions of cam-
bial activity (Moyle et al., 2002), and PttIAA3 was identified as
an important mediator of auxin-dependent regulation of cambial
proliferation activity (Nilsson et al., 2008). When a Eucalyptus
Aux/IAA member IAA4 (EgrIAA4) was ectopically expressed in
Arabidopsis, secondary xylem formation of the transgenic plants
was dramatically reduced (Yu et al., 2015).
Various plant model systems have been used to study the
mechanisms governing plant vascular development. The con-
struction of genetic networks underlying vascular tissue specifica-
tion has been mainly achieved based on research on four organs/
systems: provascular specification during embryogenesis, procam-
bial cell specification during root development (primary growth),
(pre)procambial cell specification during leaf vein patterning (pri-
mary growth), and cambium specification during secondary
growth in stem for wood formation (Campbell & Turner, 2017).
For embryogenesis and procambial specification during primary
growth in roots and leaf veins, the key components of auxin sig-
naling have been identified: the IAA12/BDL-ARF5/MP module
for embryogenesis (Hardtke & Berleth, 1998; Hamann et al.,
2002), the ARF5/MP-mediated pathway for leaf vein patterning
(Przemeck et al., 1996; Donner et al., 2009), and the IAA20/
IAA30-ARF5/MP combinations for root development (Muller
et al., 2016). To date, however, it remains unknown which Aux/
IAA-ARF combination mediates auxin-dependent cambial
specification of secondary growth for wood formation.
Here, we identified PtoIAA9 as a novel key component in
modulating wood formation in Populus tomentosa. Functional
characterization of the PtoIAA9–PtoARF5 module revealed
auxin-dependent differentiation of secondary xylem derived from
the cambium, and demonstrated their roles in orchestrating
xylem cell specification, woody cell size and vessel density. More-
over, we provided biochemical and genetic evidence that PtoHB7
and PtoHB8, encoding HD-ZIP III transcription factors, are
direct targets of the PtoIAA9–PtoARF5 module to coordinate
cellular behaviors associated with woody cell differentiation. Our
results provide novel insights into auxin-dependent regulation on
the dynamic and coordinated cellular events for secondary xylem
differentiation in woody species.
Materials and Methods
Gene cloning and plasmid construction
The full-length coding sequence of PtoIAA9,PtoARF5.1 and
PtoARF5.2 was amplified from cDNA of P. tomentosa using
gene-specific primer pairs (Table S1), and constructed into the
pCXSN vector (Chen et al., 2009), respectively. To substitute the
first proline (P) with serine (S) within the Degron motif, overlap
PCR was performed using pCXSN-PtoIAA9 as templates. The
resulting PCR product was ligated into pCXSN under control of
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the CaMV 35S promoter to generate the plasmid for overexpress-
ing PtoIAA9m. The full-length sequence of PtoARF5.1 was sub-
cloned into the modified pCAMBIA1305 with a kanamycin
resistant gene. The sequence of PtoARF5.1Dharboring the N-
terminal 1995 nucleotides of PtoARF5.1 was amplified from
pCXSN-PtoARF5.1 and constructed into the modified
pCAMBIA1305 vector. A 2-kb promoter fragment upstream of
PtoIAA9 was amplified from genomic DNA of P. tomentosa using
the primer pair Pro-PtoIAA9-fw/rv (Table S1), and inserted into
pXGUS-P to drive the GUS reporter gene (Chen et al., 2009).
Genetic transformation and growth conditions
P. tomentosa was stably transformed using the method of
Agrobacterium-mediated infiltration of leaf disks as described pre-
viously (Jia et al., 2010). PCR genotyping with the primers of
hygromycin/kanamycin-resistant genes was performed for the
identification of positive transgenic plants. Transgenic and WT
poplar plants were propagated via in vitro microcutting. For
clonal propagation, shoot segments of 3–4 cm with two or three
young leaves were cut from sterilized seedlings and cultivated on
woody plant medium solid medium at 25°C with 16 h light of
5000 lux and 8 h dark. For phenotype analyses, 4-wk-old micro-
cutting-propagated seedlings were transferred to soil in pots, and
cultivated in a glasshouse at 23–25°C with the light of 10 000 lux
under a 16 h: 8 h, day : night cycle.
Cross-sectioning and histological staining
The 7
th
internodes were sectioned with a razor blade, and then
stained with 0.05% (w/v) toluidine blue for 5 min. The cross-
sections were observed and captured using a microscope (Zeiss).
The images were analyzed using IMAGEJ (https://imagej.nih.gov/
ij/) for quantifying morphological parameters of xylem cells.
RNA in situ hybridization
For probe preparation, 276- and 223-bp gene-specific cDNA
fragments were amplified for PtoIAA9 and PtoARF5.1, respec-
tively (Table S1). The two probes were labeled using a DIG RNA
Labeling Kit (Roche). Section pretreatment, hybridization and
immunological detection were performed as previously described
(Sang et al., 2012).
GUS staining
For GUS staining (Jefferson, 1987), cross-sections of the 7
th
intern-
odes of 2-month-old plants were fixed in acetone for 1 h at 20°C,
and then washed twice in double distilled H
2
O(ddH
2
O). The
cross-sections were soaked in GUS staining solution (0.5 M Tris,
pH 7.0, 10% Triton X-100 with 1 mM X-Gluc (5-bromo-4-
chloro-3-indolyl-D-glucuronide)) for 15 min at 37°C in the dark.
After reaction, Chl was removed by use of 75% ethanol three times
at 65°C. The Chl-free stained stems were observed under an
Olympus 566 SZX16 microscope (Tokyo, Japan) and documented
using an Olympus DP73 camera.
qRT-PCR
Total RNA was extracted from tissues of 2-month-old
P. tomentosa plants using a Plant RNeasy Mini Kit (Qiagen).
cDNA was synthesized using a PrimeScript
TM
RT reagent Kit
with gDNA Eraser (Takara, Dalian, China). Quantitative real
time polymerase chain reaction (qRT-PCR) was performed using
SYBR Premix ExTaqTM (Takara) in a TP800 Real-Time PCR
machine (Takara). The poplar 18S rRNA gene was used as the
reference gene as an internal standard. The primers used for
qRT-PCR are listed in Table S1.
Transactivation test in yeast
The recombinant plasmids were introduced into the yeast strain
Saccharomyces cerevisiae Gold2 using the PEG/LiAC method.
The transformed strains were screened on synthetic dropout (SD
medium) lacking tryptophan (Trp; SD/-T) for selection of posi-
tive clones. Subsequently, positive clones were transferred to SD
medium lacking Trp, histidine (His) and adenine (Ade; SD/-
ATH) and cultivated at 28°C for 2 d. Positive clones were used
for transactivation analysis on X-a-gal indicator plates. A digital
camera (EOS 550D, Canon, Tokyo, Japan) was used to pho-
tograph yeast cells.
Yeast two-hybrid (Y2H) assays
Y2H assays were performed based on the manufacturer’s instruc-
tions (Clontech, Palo Alto, CA, USA). The AD and BD fusion
constructs were co-transformed into yeast strain Gold2 as
described above. The transformants were screened on SD
medium lacking tryptophan (Trp) and leucine (Leu; SD/-TL) at
28°C. After 3 d, positive clones were cultured in YPD liquid
medium for 1 d to an OD
600
of 1.0, and the yeast solution was
diluted with ddH
2
Oto1–10
3
x, and then transferred to SD
medium lacking Trp, Leu, histidine (His) and adenine (Ade;
SD/-AHTL) and cultured for 2 d. A EOS 550D digital camera
was used to photograph yeast cells.
Bimolecular fluorescence complementation (BiFC)
For BiFC assays, full-length coding sequences of PtoIAA9 and
PtoARF5.1 were cloned from pCXSN-PtoIAA9 and pCXSN-
PtoARF5.1, and ligated into pXY104 and pXY106 vectors,
respectively. The vectors pXY104 and pXY106 carrying the N-
and C-terminal halves of yellow fluorescent protein (YFP) were
used. The open reading frame (ORF) of PtoIAA9 was cloned into
pXY106, while that of PtoARF5.1 was constructed into pXY104.
The constructs were co-transformed into tobacco as described
above, and observed by confocal laser microscopy (Olympus 589
FV1200).
Chromatin immunoprecipitation (ChIP) assay
ChIP analysis was performed as previously described (Yang et al.,
2012). One-month-old PtoARF5.1 transgenic poplar plants
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harboring the HA epitope were used as samples; the HA antibody
and normal mouse IgG were used for immunoreaction. All
primers used in ChIP assays are listed in Table S1.
Effector-reporter test
Promoters of the PtoHB7 and PtoHB8 genes were amplified via
specific primers (Table S1) and constructed into pCXGUS-P.
The 35S:PtoARF5.1 and 35S:PtoARF5.1Dvectors were used as
effectors. Tobacco leaves were transformed by Agrobacteria con-
taining the effectors and reporters as described above. After 3 d of
infiltration, GUS activity was measured by monitoring the cleav-
age of 4-methyl umbelliferyl b-D-glucuronide (MUG), the sub-
strate of b-glucuronidase, which produces the fluorescent
4-methyl umbelliferone (4MU) upon hydrolysis. Protein concen-
trations were determined via the Bradford method.
Gene accessions
GeneBank accession numbers of the P. tomentosa genes are:
PtoIAA9 (MH345700), PtoARF5.1 (MH352401), PtoARF5.2
(MH352402) and PtoHB7 (MH345699).
Results
IAA9 is highly expressed across wood-forming tissues in
poplar
A total of 35 Aux/IAA genes were previously predicted in the
poplar genome (Kalluri et al., 2007). These Aux/IAA genes were
identified in the more recent release of the Populus trichocarpa
genome (v3.0; https://phytozome.jgi.doe.gov) and designated
according to phylogenetic relationships with their Arabidopsis
orthologues (Fig. S1a). To determine the members that play a
key role during wood formation, expression levels of all Aux/IAA
genes were comprehensively evaluated using the high-spatial-
resolution RNA sequencing data of the wood-forming tissues in
poplar (Sundell et al., 2017). Among them, IAA9,-11,-16.1,-
16.2 and -29.1 showed high expression (Fig. S1). Continuous
assays across secondary xylem tissues in poplar stems (Sundell
et al., 2017) allowed us to evaluate spatial expression patterns of
these highly expressed Aux/IAAs and some other members, such
as IAA12.1,-12.2,-20.1 and -20.2 (Fig. S2b). A highly similar
expression pattern was shared by IAA9,-16.1 and -16.2, extend-
ing from the cambium to differentiated xylem (Fig. S2b), which
was validated by correlation tests (Pearson correlation coefficient
R
2
=0.82 or 0.51, P<0.01; Fig. S2c). For this pattern, the tran-
scripts of these three Aux/IAAs accumulated in the cambial zone
and developing xylem, and then decreased towards differentiated
xylem (Fig. S2d), consistent with the previously reported auxin
distribution across wood-forming tissues of poplar stems (Uggla
et al., 1998; Immanen et al., 2016). By contrast, IAA11 and -29.1
displayed totally different expression patterns (Fig. S2b). Thus,
IAA9,-16.1 and -16.2 were considered key Aux/IAA genes of
auxin signaling involved in wood formation in poplar. Tissue-
specific expression assays by qRT-PCR in P. tomentosa further
confirmed the enriched transcript abundance of IAA9,-16.1 and
-16.2 in stems (Fig. S2d). We selected PtoIAA9 from P. tomentosa
as a candidate gene to further investigate auxin-dependent regula-
tion of wood formation.
To determine the exact expression pattern of PtoIAA9 in sec-
ondary vascular tissues, RNA in situ hybridization was performed
using the fifth internode of 1.5-month-old poplar (Fig. 1a,b).
Transcripts of PtoIAA9 were preferentially accumulated in the
cambial zone and neighboring cells (Fig. 1a). Furthermore, the
GUS reporter driven by the PtoIAA9 promoter in transgenic
poplar confirmed its expression in the cambium zone and closely
neighboring cell layers of the wood-developing stem (Fig. 1c,d).
qRT-PCR assays revealed a more than 3-fold induced transcript
abundance of PtoIAA9 in stems within 3 h of exogenous auxin
(Fig. 1e). The auxin-inducible expression was also examined by
quantification of GUS activity driven by the PtoIAA9 promoter
in wood-forming tissues (Fig. 1f). These results indicated that
PtoIAA9 is an auxin-inducible gene predominantly expressed in
cambium and adjacent cells towards xylem differentiation.
PtoIAA9 encodes a canonical Aux/IAA protein belonging
to a distinct clade
PtoIAA9 protein harbors the characteristic domains (Domain I,
II, III and IV; Reed, 2001) of Aux/IAAs in high sequence similar-
ity to its close homologs in other plant species (Fig. S3). Remark-
ably, PtoIAA9 belongs to a distinct clade among all Aux/IAA
members due to its 50% longer amino acid sequence (c. 300 aa)
compared with the average length (c. 200 aa) of other Aux/IAA
members (Wang et al., 2005). Transient expression of a PtoIAA9-
GFP fusion gene revealed that PtoIAA9 is a nucleus-localized
protein (Fig. S4a; Methods S1), consistent with the prediction of
both bipartite and SV40-type nuclear localization signals (NLS)
present within its sequences (Fig. S3). Aux/IAA proteins usually
contain a short amino acid stretch (VGWPP) called Degron that
confers auxin-stimulated protein turnover (Worley et al., 2000).
Due to the presence of a typical Degron motif in its Domain II,
auxin-responsive protein stability of PtoIAA9 fused with a green
fluorescent protein (GFP) tag was monitored in transiently
expressed epidermal cells of tobacco leaves (Fig. S4b; Meth-
ods S1). The fluorescent signals of PtoIAA9-GFP were signifi-
cantly reduced in the leaf epidermal cells subject to IAA
(Fig. S4b), as validated by quantifying the percentage of fluores-
cent nuclei and fluorescence intensity per nucleus (Fig. S4c,d).
By contrast, this auxin-induced protein instability disappeared
when the first proline (P) within the Degron motif of PtoIAA9
was replaced by serine (S) (ns, not significant; P>0.05; Fig. S4b–
e). Moreover, stronger fluorescence was detected in these leaf epi-
dermal cells transiently transformed by the PtoIAA9 mutant gene
with impaired Degron motif, compared to the wild-type control
(Fig. S4b–d), indicating that the auxin-inducible rapid turnover
of PtoIAA9 depends on its Degron motif. Self-activation assays
in yeast showed that PtoIAA9 has no transcriptional activating
activity (Fig. S4f). Additionally, PtoIAA9 compromised the acti-
vating capability of the VP16 domain, demonstrating that it is a
transcriptional repressor. The truncated PtoIAA9 protein without
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Domain I at the C-terminal region (BD-VP16-PtoIAA9t)
showed repressing activity (Fig. S4f ). Taking this together,
PtoIAA9 exhibits molecular features typical of canonical Aux/
IAA repressors.
Overexpression of an auxin-resistant PtoIAA9m represses
wood formation
To identify its role in regulating secondary xylem development
during wood formation, the auxin-resistant PtoIAA9 containing
the impaired Degron sequence (Fig. S4e; PtoIAA9m) was consti-
tutively expressed in poplar (PtoIAA9m-OE; Fig. S5a). Represen-
tative overexpressing lines displayed significantly reduced plant
growth compared to the wildtype (WT; Fig. 2a). Quantitative
measurement revealed that overexpression of PtoIAA9m led to a
40–50% reduction in plant height and 35% reduction in stem
diameter (Fig. 2b,c). Similar changes were indicated by a time-
course analysis of these two parameters (Fig. S5b,c). The intern-
ode number of WT and transgenic plants was not changed (ns;
P>0.05; Fig. S5d). Secondary vascular tissues were examined in
PtoIAA9m-OE plants stained with toluidine O (Fig. 2d).
Secondary xylem development was significantly repressed by
overexpressing PtoIAA9m (Fig. 2d), with a 40% decrease in the
number of xylem cell layers, compared with WT (Fig. 2e). The
percentage of secondary xylem occupying the whole stem was
46.8% in WT, but was attenuated to 39.1% in the PtoIAA9m-
OE lines, whereas that of phloem was not affected (Fig. 2f).
These results indicated that PtoIAA9m might inhibit wood
formation.
PtoIAA9-attenuated auxin signaling impairs secondary
xylem formation
To determine PtoIAA9-dependent regulation of wood forma-
tion, we first examined phenotypes of wood-associated cell types,
including cambium, xylem fibers and vessels in transgenic poplar.
The results showed that cambial cells were arrayed more tightly
in PtoIAA9m-OE plants than WT (Fig. 2g), but the number of
cambial cell layers was not significantly affected by overexpression
of PtoIAA9m (ns; P>0.05; Fig. S5e). Cross-sections of different
internodes in WT revealed the presence of one or two particular
cell layers on the side of the cambium zone towards the xylem
(a) (b) (e)
(f)
(c) (d)
Fig. 1 Expression pattern and auxin induction of PtoIAA9 transcripts in wood-forming tissues of Populus tomentosa stem. (a, b) RNA in situ hybridization
of PtoIAA9 in secondary vascular tissues of poplar. The 5
th
internodes of 1.5-month-old poplar plants cultivated in soil were cross-sectioned for
hybridization with antisense (a) and sense (b) probes of PtoIAA9. Red triangles indicate in situ hybridization signals for PtoIAA9 transcripts. Ca, cambium;
Ph, phloem; Pi, pith; Xy, xylem. (c, d) Histological staining of the GUS reporter driven by the promoter of PtoIAA9 in poplar stems. The 7
th
internodes of
1.5-month-old poplar plants cultivated in soil were cross-sectioned for GUS staining. Ca, cambium; Ph, phloem; Xy, xylem.. (e) Time-course assays of
auxin-induced transcript abundance of PtoIAA9 in poplar stems. The microcutting-propagated poplar seedlings cultivated in vitro for 4 wk were subjected
to 5 lM IAA for 0, 1, 2 and 3 h, and stem tissues were collected for RNA extraction followed by qRT-PCR assays. 18S rRNA was used as a reference gene.
Expression levels are indicated relative to values for 0 h (with 0 h set arbitrarily to 1). Error bars represent SD. Asterisks indicate significant differences
between mock and auxin treatment at each time point (Student’s ttest): *,P<0.05; **,P<0.01; ***,P<0.001; n=3. (f) Quantification of auxin-induced
GUS activity driven by the promoter of PtoIAA9 in poplar stem. IAA treatment (5 lM) was performed for 6 h as indicated in (d). The values under mock
treatment were normalized to 1. Error bars represent SD. Asterisks indicate significant differences with respect to mock (Student’s ttest): **,P<0.01;
n=4. Bars: (a, b) 200 lm; (c) 500 lm; (d) 100 lm.
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(Figs 2g, S5f). These cells were not stained by toluidine blue, sug-
gesting that lignin deposition was not initiated for secondary cell
wall formation. These cells were distinctly larger than cambial
cells but smaller than mature xylem cells (Figs 2g, S5f). These fea-
tures indicated that these cells were newly differentiated from
cambium zone towards xylem and undergoing cell expansion,
and thereby considered as early developing xylem (EDX).
However, these EDX cell layers were greatly reduced and almost
absent in cross-sections of PtoIAA9m-OE stems (Fig. 2g), resem-
bling EDX cells just before dormancy due to reduced rate of cam-
bium cell periclinal division. This greatly reduced EDX cell layer
suggests that overexpression of stabilized PtoIAA9 inhibited the
periclinal division of cambium, thus leading to reduced wood
formation.
(a) (d)
(b) (c) (e) (f)
(g) (h) (i) (j)
Fig. 2 Wood phenotypes resulting from PtoIAA9m overexpression in Populus tomentosa. (a) Dwarf phenotypes of 2-month-old plants of independent
PtoIAA9m-overexpressing (PtoIAA9m-OE) transgenic poplar lines (L1 and L2). (b, c) Measurement of plant height (b) and stem diameter (c) of PtoIAA9m-
OE transgenic poplar lines corresponding to (a). (d) Cross-sectioning and staining with toluidine blue of the 7
th
internode of 2-month-old wild-type (WT)
and PtoIAA9m-OE transgenic plants (L1). Ph, phloem; Pf, phloem fibers; Xy, xylem. (e) Quantification of secondary xylem cell layers in WT and
PtoIAA9m-OE transgenic plants (L1). The number of secondary xylem cell layers was counted in toluidine blue-stained anatomical sections of the 7
th
internode of WT and PtoIAA9m-OE transgenic plants. (f) Percentage of secondary xylem and bark in the stem of WT and PtoIAA9m-OE transgenic plants
(L1). The area of secondary xylem, bark and total stem was measured via IMAGEJ in toluidine blue-stained anatomical sections of the 7
th
internode of WT
and PtoIAA9m-OE transgenic plants. (g) Detailed observation of the cambial zone and woody cells of secondary xylem in WT and PtoIAA9m-OE plants.
The images were captured on toluidine blue-stained anatomical sections of the 7
th
internode of the corresponding lines. White lines indicate cambium (Ca),
and red stars represent the cells of early developing xylem (EDX). V, vessel. (h, i) Quantification of the size of a single fiber (h) and vessel (i) cell in stem of
WT and PtoIAA9m-OE plants. The area of fiber and vessel cells was measured and calculated via IMAGEJ based on the images of toluidine blue-stained
anatomical sections as described in the Materials and Methods section. (j) Density of vessels in stem of WT and PtoIAA9m-OE plants. The number of
vessels was counted based on the images of toluidine blue-stained anatomical sections. Error bars represent SD. Asterisks indicate significant differences
with respect to values of WT (Student t-test): *,P<0.05; **,P<0.01; ***,P<0.001; n=4. Bars: (a) 5 cm; (d) upper, 250 lm, lower, 100 lm; (g) 50 lm.
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Quantitative measurement showed that the cell size of xylem
fibers and vessels was reduced by 36% and 28% in PtoIAA9m-
OE plants relative to WT (Fig. 2g–i). By contrast, vessel density
was significantly increased by 42% in PtoIAA9m-OE plants
(Fig. 2g,j). Cell wall thickness was not affected by overexpressing
PtoIAA9m (ns; P>0.05; Fig. S5g). Together, these data sug-
gested that PtoIAA9m inhibited developing xylem cell (vessel
and fiber) differentiation and expansion.
PtoIAA9 interacts with PtoARF5s
PtoIAA9 contains intact Domains III and IV (also called PB1
domain) that mediate physical interaction with ARFs, a family of
plant-specific B3-type transcription factors directly driving the
expression of auxin responsive genes (Vanneste & Friml, 2009).
We speculated that PtoIAA9 might interact with ARF proteins to
regulate wood formation in poplar. A previous study has shown
that ARF5/MP is a key regulator of auxin-dependent vascular
patterning during embryogenesis and leaf vein patterning in Ara-
bidopsis (Donner et al., 2009; Brackmann et al., 2018). The
poplar genome harbors duplicated ARF5 members, designated
ARF5.1 and ARF5.2, which are expressed in secondary vascular
tissues of stems (Johnson & Douglas, 2007). Poplar ARF5s share
conserved modular structure with their Arabidopsis homolog
containing intact Domains III and IV (Fig. S6a), implying that
they may interact with PtoIAA9. To determine the interactions
between PtoIAA9 and PtoARF5s, we performed Y2H assay
(Fig. 3a). Owing to the presence of the B3 domain with DNA-
binding activity, the coding region of PtoARF5s was fused to
AD, and the resulting fused protein was subsequently coexpressed
in yeast cells with PtoIAA9-BD. Both PtoARF5 paralogs were
shown to interact with PtoIAA9, but PtoARF5.1 displayed
stronger interaction ability than PtoARF5.2 (Fig. 3a). The
PtoIAA9–PtoARF5 interaction was subsequently confirmed by
BiFC analyses (Fig. 3b).
Despite the physical interactions, the PtoIAA9–PtoARF5-
dependent regulation on wood formation requires their coexpres-
sion in secondary vascular tissues. Analysis of expression patterns
based on RNAseq datasets (Sundell et al., 2017) revealed the
highly correlated transcript accumulation between IAA9 and
ARF5s in poplar wood-forming tissues (Fig. S6b,c). Similar to
the expression patterns of PtoIAA9, RNA in situ hybridization
results showed that PtoARF5.1 was highly expressed in develop-
ing wood-associated tissues of poplar (Fig. 3c,d).
PtoARF5.1 rescues PtoIAA9m-affected secondary xylem
differentiation
Physical interactions and overlapping expression of PtoIAA9 and
PtoARF5s suggested their cooperative involvement in the
regulation of wood formation. To test this hypothesis, functional
complementation of PtoARF5.1 was performed on PtoIAA9m-
OE-resulting phenotypes of secondary xylem development
(Fig. 4). To avoid the undesirable effects of PtoIAA9 overexpres-
sion and other endogenous Aux/IAA genes on its function, a full-
length PtoARF5.1 and a truncated form without C-terminal
Domains III and IV responsible for Aux/IAA interactions
(PtoARF5.1D) were introduced into the PtoIAA9m-OE trans-
genic lines, respectively (Figs 4, S6). Y2H tests showed that the
C-terminal deletion abolished the interactions of PtoARF5.1
with the PtoIAA9 protein (Fig. S7a), but did not affect transcrip-
tional activation (Fig. S7b).
Phenotypic analysis of transgenic plants showed that reduced
growth of the PtoIAA9m-OE lines could be partially rescued by
PtoARF5.1, whereas the truncated PtoARF5.1 displayed stronger
recovery than the full-length form (Fig. 4a). Quantitative mea-
surement of plant height and stem diameter confirmed these
changes in different transgenic lines (Fig. 4b,c). By contrast with
the partial recovery (c. 50%) by the full-length PtoARF5.1, the
PtoARF5.1Dalmost completely rescued the decreased stem diam-
eter of the PtoIAA9m-OE plants relative to that of WT (Figs 4c,
S7d). Stem cross-sectioning was conducted to establish if rescue
of secondary xylem growth by constitutive expression of
PtoARF5.1 occurred in the PtoIAA9m-OE lines (Fig. 4d–f). Both
the full-length and the truncated PtoARF5.1 were able to rescue
the inhibition of overexpressing PtoIAA9m on secondary xylem
development, as evidenced by the increased number of xylem cell
layers and xylem percentage (40% by PtoARF5.1 and 94% by
PtoARF5.1D) in stems (Fig. 4d–f). Noticeably, PtoARF5.1Dcon-
ferred stronger recovery of the PtoIAA9m-OE-resulting defective
wood formation than the full-length PtoARF5.1 (Fig. 4d–f), lead-
ing to even more xylem cell layers than WT.
Subsequently, the cambium zone and its adjacent cell layers
were characterized in detail to reveal the possible modulation of
cell differentiation by the PtoIAA9–PtoARF5 module (Fig. 5).
By contrast with the absence of EDX in PtoIAA9m-OE plants,
these cell layers reoccurred in both PtoARF5.1 and PtoARF5.1D-
complementing lines (Fig. 5a). Similar recovery of defective
xylem cell expansion resulting from overexpression of PtoIAA9m
was detected for PtoARF5.1 and PtoARF5.1D, as indicated by
measurement of fiber and vessel cell size (Fig. 5b–d). Moreover,
elevated vessel density was also rescued by introduction of
PtoARF5.1 and PtoARF5.1Dinto the PtoIAA9m-OE lines
(Fig. 5b,e). The stronger recovery of PtoARF5.1Dthan its full-
length form indicated that the PB1 domain mediating AUX/
IAA-ARF protein interactions compromises the rescue of
PtoIAA9m-resulting phenotypes of secondary xylem differentia-
tion. Therefore, PtoARF5 is able to drive the PtoIAA9-
dependent cellular behaviors for secondary xylem differentiation
in poplar.
The PtoIAA9–PtoARF5 module directly regulates
expression of PtoHB7/8
Diverse auxin-triggered developmental phenotypes depend on
expression of specific batteries of auxin responsive genes targeted
by Aux/IAA-ARF pairs (Vanneste & Friml, 2009). To identify
auxin responsive genes targeted by the PtoIAA9–PtoARF5
module during wood formation in poplar, comparative transcrip-
tomic profiling via RNAseq was performed with the PtoIAA9m-
OE lines and WT, revealing 3873 differentially expressed genes
(fold change ≥2 and false discovery rate ≤1%; Fig. S8a;
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Methods S1; Dataset S1). A number of Aux/IAAs, SAURs and
GH3s, which fall into classic families of early auxin responsive
genes in plants (Guilfoyle, 1999), displayed significantly reduced
transcript abundance (2.1- to 15.2-fold) in the PtoIAA9m-OE
lines (Fig. S7b), suggesting that auxin signaling was weakened by
PtoIAA9m overexpression in poplar. Gene ontology (GO) analy-
sis of differentially expressed genes showed enrichment of the
term of nucleic acid binding transcription factor activity
(Fig. S7c). We thus investigated the differentially expressed genes
encoding transcription factors that are known to regulate wood
formation in poplar (Fig. S7d). All of these genes, including
WNDs, ANT and HBs, displayed decreased expression in the
PtoIAA9m-OE lines (Fig. S8d). It has previously been established
that class III HD-ZIP transcription factors are critical regulators
of vascular formation and patterning in plants (Ramachandran
et al., 2017). This family, designated HB transcription factors,
comprises eight members in poplar (Fig. S8e). RNAseq and
qRT-PCR showed that five of them displayed PtoIAA9m-
inhibited expression (Fig. S8d,f). Among them, PtoHB7 and
PtoHB8 were identified as critical regulators of vascular cambium
differentiation to secondary xylem in poplar (Zhu et al., 2013).
Therefore, we investigated whether the PtoHB7/8genes are
downstream targets of PtoIAA9–PtoARF5 during wood forma-
tion.
To test this hypothesis, we determined the regulation of
PtoIAA9–PtoARF5 on PtoHB7 in the PtoIAA9m-OE lines. As
(a)
(b) (c)
(d)
Fig. 3 Protein interactions of PtoIAA9 with PtoARF5s and expression of PtoARF5 in secondary vascular tissues of Populus tomentosa stem. (a) Yeast-two-
hybrid analysis of protein interactions between PtoIAA9 and PtoARF5.1/5.2. PtoIAA9 and PtoARF5.1/5.2 were fused with a Gal4 DNA-binding domain
(BD) and a GAL4 activation domain (AD), respectively. The interaction between BD-p53 and AD-RecT (SV40 large T-antigen) was used as a positive
control, while those between blank constructs (BD or AD) with BD-PtoIAA9 or AD-PtoARF5.1/5.2 were used as negative controls. Yeast cells were
inoculated on selective medium in a 10-fold gradient dilution. SD/-TL, double dropout medium lacking tryptophan and leucine; SD/-AHTL, quadruple
dropout medium lacking adenine, histidine, tryptophan and leucine. (b) Bimolecular fluorescence complementation (BiFC) assays validating physical
interactions between PtoIAA9 and PtoARF5 in nuclei. nYFP and cYFP represent the N- and C-terminal part of yellow fluorescent protein (YFP),
respectively. The PtoIAA9-nYFP and PtoARF5.1-cYFP constructs were cotransfected into tobacco epidermal leaf cells via Agrobacterium-mediated
infiltration. The blank constructs of cYFP or nYFP were cotransfected with PtoIAA9-nYFP or PtoARF5.1-cYFP, respectively, as negative controls. The
fluorescence emitted by YFP was examined with a confocal microscope. Nuclei were identified by DAPI staining. (c, d) RNA in situ hybridization of
PtoARF5 in secondary vascular tissues of poplar stem. The 7
th
internodes of 6-wk-old poplar plants cultivated in soil were cross-sectioned for hybridization
with sense (c) and anti-sense (d) probes. The probes were designed within the identical region of PtoARF5.1 and PtoARF5.2 for detection of both ARF5
paralogs in poplar. Red triangles indicate the in situ hybridization signals. Ca, cambium; Ph, phloem; Pi, pith; Xy, xylem. Bars: (b) 50 lm; (c, d) 200 lm.
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shown in Fig. 6(a), the expression of both PtoHBs was signifi-
cantly decreased by 71% and 74% in the PtoIAA9m-OE lines,
respectively, and could be rescued by the introduction of the
truncated PtoARF5.1 to even higher levels than that of WT. We
also found that expression of PtoHB7 and PtoHB8 was increased
3.9- and 1.9-fold, respectively, in response to auxin treatment
(Fig. 6b). Noticeably, overexpression of PtoIAA9 almost com-
pletely blocked the auxin-induced expression of both PtoHB7
and PtoHB8 in the wood-forming stem (Fig. 6b), indicating that
the auxin responsive expression of PtoHB7 and PtoHB8 is depen-
dent on PtoIAA9. Promoter sequence analysis revealed that a
series of core and canonical auxin response elements (AuxREs),
potential DNA binding sites for ARFs, are predicted in the 1.5 kb
upstream regions of the start codons of PtoHB7 and PtoHB8
(Fig. 6c,d). ChIP was performed using PtoARF5.1-HA transgenic
plants and the enrichment of AuxREs harbored by the PtoHB7
and PtoHB8 promoters was quantified by qPCR (Fig. 6e,f). In
comparison with negative controls, some AuxRE-containing pro-
moter regions of PtoHB7 (Regions II/III) and PtoHB8 (Regions
III/IV) were significantly enriched after immunoprecipitation
(Fig. 6e,f), indicating the direct binding of PtoARF5.1 to their pro-
moters. The PtoIAA9/PtoARF5-dependent regulation of PtoHB7/8
was subsequently confirmed by effector–reporter assays in transiently
expressed tobacco leaves. The reporter constructs carrying the GUS
reporter gene driven by the 1.5 kb promoter regions of PtoHB7 and
PtoHB8, respectively, were co-transfected with different combina-
tions of the effectors harboring PtoIAA9 and PtoARF5.1 (Fig. S9).
Determination of GUS activity revealed that PtoARF5.1 was able to
independently activate expression of PtoHB7, whereas the activation
was completely abolished by the addition of PtoIAA9 (P<0.05;
Fig. S9a). By contrast, PtoIAA9 did not compromise the activation
induced by PtoARF5.1Dwithout the C-terminal III/IV domains,
indicating that PtoIAA9-mediated repression of PtoHB7 expression
relies on its interactions with PtoARF5.1 via the III/V domains.
Similar results were obtained from the promoter of PtoHB8
(Fig. S9b).
To further validate direct regulation of PtoARF5.1 on PtoHBs
via binding to AuxREs, short promoter fragments harboring
(a) (b)
(d) (e)
(f)
(c)
Fig. 4 Rescue of PtoIAA9m-resulting wood-associated phenotypes in Populus tomentosa by PtoARF5. (a) Recovered morphological phenotypes of 1.5-
month-old PtoIAA9m-overexpressing (OE) transgenic poplar plants by PtoARF5. The constructs of 35S:PtoARF5.1 and 35S:PtoARF5.1D(encoding for 1–
665 amino acids of PtoARF5.1 without C-terminal III/IV domains) were transformed into poplar in the background of PtoIAA9m-OE. (b, c) Measurement
of plant height and stem diameter of wild-type (WT), PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE transgenic poplar
lines. (d) Anatomical sections stained with toluidine blue of the 7
th
internode of 1.5-month-old WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and
PtoARF5.1D-OE/PtoIAA9m-OE plants. Red lines indicate xylem. Xy, xylem. (e) Quantification of secondary xylem cell layers of WT, PtoIAA9m-OE,
PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE lines. The number of xylem cell layers was counted in toluidine blue-stained anatomical
sections of the 7
th
internode of WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. (f) Percentage of
secondary xylem and bark in the stem of WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. The area of
secondary xylem, bark and total stem was quantified via IMAGEJ in toluidine blue-stained cross-sections of the 7
th
internode of WT, PtoIAA9m-OE,
PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. Error bars represent SD. The letters above error bars indicate statistically
significant differences (one-way ANOVA followed by Dunnett’s test for pairwise comparisons; n=4). Bars: (a) 5 cm; (d) 100 lm.
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canonical AuxREs(447 to 581 bp upstream of the start codon
of PtoHB7, and 344 to 434 bp upstream of the start codon of
PtoHB8), which were strongly enriched in ChIP assays, were con-
structed to drive the firefly luciferase reporter. Consistently,
PtoARF5.1 significantly activated the expression of luciferase
reporter driven by the AuxRE-harbored promoter fragments of
PtoHB7/8(P<0.05; Fig. 6g). By contrast, PtoARF5.1-driven
activation disappeared when the AuxREs harbored in the pro-
moter fragments of PtoHB7 and PtoHB8 were disrupted by site-
directed mutagenesis (Fig. 6g). Therefore, the PtoIAA9–
PtoARF5 module directly regulates the expression of PtoHB7
and PtoHB8 via binding of PtoARF5 to the AuxREs within their
promoters.
PtoHB7 partially rescues PtoIAA9m-resulting phenotypes
of wood formation
To establish the link of biological functions between auxin signal-
ing and the HB transcription factors during wood formation, we
conducted complementation of the PtoIAA9m-resulting impaired
secondary xylem phenotypes by PtoHB7 via stable transformation
(Figs 7, S9). Phenotypic characterization revealed partial recovery
of the PtoIAA9m-repressing plant growth and stem development
by PtoHB7 (Fig. S10b–d). Stem cross-sections showed that intro-
duction of PtoHB7 into the PtoIAA9m-OE background could
partially rescue the impaired secondary xylem development, as
validated by the quantification of xylem cell layers (Fig. 7a,b).
Constitutive expression of PtoHB7 led to the reoccurrence of the
EDX cell layers, which were repressed in the PtoIAA9m-OE lines
(Fig. 7c). Similarly, the decreased cell size of xylem fibers and ves-
sels as well as the enhanced vessel density were partially rescued
by the introduction of PtoHB7 into the PtoIAA9m-OE plants
(Fig. 7d–h).
Moreover, we determined the expression of several genes,
including ACL5,CesA7A,PAL4,GT43B and WND1B, which
were previously reported HB7/8-regulated genes involved in
wood formation of poplar (Milhinhos et al., 2013; Zhu et al.,
2013). The down-regulated expression of these genes in
PtoIAA9m-OE plants was at least partially rescued in PtoARF5.1-
and PtoHB7-complementing transgenic lines (Fig. S11). A simi-
lar result was obtained in EXPA1, a marker gene for cell expan-
sion during wood formation (Sundell et al., 2017). We also
(a) (b)
(c) (d) (e)
Fig. 5 PtoARF5 restores PtoIAA9-affected phenotypes of secondary xylem cells in Populus tomentosa during wood formation. (a, b) Detailed observation
of cambial zone and woody cells of secondary xylem in wild-type (WT), PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-
OE plants. The images were captured on toluidine blue-stained anatomical sections of the 7
th
internode of the corresponding lines. White lines indicate
cambium (Ca), and red stars represent the cells of early developing xylem (EDX). V, vessel. Bars, 50 lm. (c, d) Quantification of size of a single fiber and
vessel cell in stems of WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. The area of fiber and vessel cells was
measured and calculated via IMAGEJ based on images of toluidine blue-stained anatomical sections as described in the Materials and Methods. (e) Density of
vessels in stems of WT, PtoIAA9m-OE, PtoARF5.1-OE/PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE plants. The number of xylem vessels was
counted based on images of toluidine blue-stained anatomical sections. Error bars represent SD. The letters above error bars indicate significant differences
(one-way ANOVA followed by Dunnett’s test for pairwise comparisons; n=10–12).
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(a) (b)
(c) (d)
(e)
(g)
(f)
Fig. 6 Direct regulation of PtoIAA9/ARF5 on PtoHB7 and PtoHB8 in Populus tomentosa. (a) Expression levels of PtoHB7 and PtoHB8 in wild-type (WT),
PtoIAA9m-OE and PtoARF5.1D-OE/PtoIAA9m-OE lines determined by qRT-PCR. Stem tissues of 6-wk-old poplar plants cultivated in soil were collected
for RNA extraction. 18S rRNA was used as a reference gene. WT values were normalized to 1. (b) Auxin-induced expression of PtoHB7 and PtoHB8 in WT
and PtoIAA9m-OE lines. Microcutting-propagated poplar seedlings cultivated in vitro for 4 wk were subjected to 5 lM IAA for 6 h, and stem tissues were
collected for RNA extraction followed by qRT-PCR assays. 18S rRNA was used as a reference gene. The values for mock treatment were normalized to 1.
For (a, b), error bars represent SD, and one-way ANOVA followed by Dunnett’s test for pairwise comparisons with respect to values of WT (a) or mock
treatment (b) was performed to detect statistically significant differences (**,P<0.001; ***,P<0.001; n=4). (c, d) Distribution of canonical and core
auxin response elements (AuxREs) harbored in the promoter regions of PtoHB7 (c) and PtoHB8 (d). Orange lines with I–III indicate AuxRE-harbored
fragments amplified by ChIP-qPCR, while NC represents AuxRE-free negative control. (e, f) ChIP-qPCR analysis of PtoARF5 protein fused with HA tag
with the promoter region of PtoHB7 (e) and PtoHB8 (f). Shoot tissues of 1-month-old poplar plants were used. Error bars represent SD. Student’s t-test
was performed to evaluate significant differences between values of WT and those of PtoARF5.1-HA for each region (**,P<0.01; ***,P<0.001; n=3).
(g) AuxRE-dependent transactivation via transient cotransformation assay. For reporters, the 1-kb promoter fragments of PtoHB7 and PtoHB8 were
constructed to drive the expression of firefly luciferase (LUC). The AuxREs found in these promoter fragments were disrupted via site-directed mutagenesis
to generate PtoHB7/8pro-m-LUC. The effector encodes PtoARF5.1 driven by the CaMV 35S promoter. Ratios of firefly luciferase to Renilla luciferase
activity after cotransformation into tobacco leaf epidermal cells with different reporter and effector construct combinations were tested. Values for the
blank effector were normalized to 1. Error bars represent SD. Student’s t-test was performed to evaluate significant differences between values of blank
effector and those of PtoARF5.1 (*,P<0.05; **,P<0.01; n=3).
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found that expression of WND6A and B, which are the closest
homologs of Arabidopsis VND6 and VND7 (Zhong et al., 2011),
was enhanced by PtoIAA9m overexpression, but was rescued by
PtoARF5.1 and PtoHB7 (Fig. S11), in accordance with elevated
vessel formation by PtoIAA9m-OE and complemented by
PtoARF5 and PtoHB7. These results strongly suggest that auxin-
induced secondary xylem development depends on the activation
of the PtoHB genes by the PtoIAA9/PtoARF5 complex in poplar.
Discussion
Auxin is considered a positional signal that drives cambium-
derived wood formation (Uggla et al., 1996). Previous studies
have demonstrated auxin-mediated regulation of cambial cell
division in trees (Sundberg et al., 2000; Nilsson et al., 2008).
However, a highly sensitive analysis of auxin signaling in Ara-
bidopsis revealed its maximal levels in differentiating cambial
descendants, spatially divergent from the maximum auxin con-
centration in cambial initials (Brackmann et al., 2018). In this
study, we report that auxin coordinates multiple aspects of the
cellular changes occurring for cambium specification into sec-
ondary xylem through the IAA9-ARF5 pathway in Populus.
We further demonstrate that the PtoIAA9–PtoARF5 module
directly targets PtoHB7 and PtoHB8 to mediate auxin-triggered
early developing xylem (EDX) cell differentiation during wood
formation.
(a)
(b) (c) (d)
(e) (f) (g)
Fig. 7 PtoHB7 partially rescues deficient phenotypes of wood formation resulting from constitutive expression of PtoIAA9 in Populus tomentosa. (a) Cross-
sections stained with toluidine blue of the 7
th
internode of 1-month-old WT, PtoIAA9m-OE and PtoHB7-OE/PtoIAA9m-OE lines. Red lines indicate xylem.
Xy, xylem. (b) Number of xylem cell layers of wild-type (WT), PtoIAA9m-OE and PtoHB7-OE/PtoIAA9m-OE lines. (c, d) Detailed phenotypes of cambial
zone and woody cells of secondary xylem in wood-forming stem of WT, PtoIAA9m-OE and PtoHB7/PtoIAA9m-OE plants. The images were captured on
toluidine blue-stained anatomical sections of the 7
th
internode of the corresponding lines. White lines indicate cambium (Ca), and red stars represent the
cells of early developing xylem (EDX). V, vessel. (e, f) Quantification of size of a single fiber and vessel cell in stems of WT, PtoIAA9m-OE, and PtoHB7/
PtoIAA9m-OE plants. The area of fiber and vessel cells was measured and calculated via IMAGEJ based on images of toluidine blue-stained anatomical
sections as described in the Materials and Methods. (g) Density of vessels in stem of WT, PtoIAA9m-OE and PtoHB7/PtoIAA9m-OE plants. The number of
xylem vessels was counted based on the images of toluidine blue-stained anatomical sections. Error bars represent SD. The letters above error bars indicate
significant differences (one-way ANOVA followed by Dunnett’s test for pairwise comparisons; n=10–12). Bars: (a) 100 lm; (c, d) 50 lm.
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Aux/IAA proteins function as molecular switches of auxin sig-
naling conserved among flowering plants (Paponov et al., 2009).
Comprehensive analyses of Populus Aux/IAAs revealed variable
levels and spatial patterns of expression across wood-forming tis-
sues (Fig. S2), implying functional diversification in wood forma-
tion (Moyle et al., 2002; Kalluri et al., 2007). The transcripts of
IAA9,-16.1 and -16.2 were enriched in cambium-neighboring
cells undergoing differentiation into xylem but declined with
xylem maturation (Fig. S2c). Highly correlated expression pat-
terns among these Aux/IAA genes (Fig. S2c,d) and common pro-
tein interactions with ARF5 (Fig. S6d) suggested their functional
redundancy in wood formation. Although compromised, wood
differentiation could not be completely suspended by overex-
pressing PtoIAA9, implying regulatory complexity depending on
some other pathways besides auxin signaling.
Compromised auxin signaling by expressing stable Aux/IAAs
allows auxin-mediated coordination of cellular behaviors during
wood formation to be explored. Overexpression of a hybrid aspen
IAA3 (PttIAA3) attenuates periclinal cell division in cambium but
enlarges cell files harboring anticlinal cell division, suggesting dual
regulation of auxin signaling on cambial proliferation (Nilsson
et al., 2008). Despite more tightly arranged cambial cells (Fig. 2g),
however, overexpressing PtoIAA9 did not lead to significant
changes in cambial cell layers (Fig. S5e), implicating its weak role
in cambial activity. IAA20.1 and -20.2, the PttIAA3 homologs in
our nomenclature, displayed much lower expression levels than
IAA9, and different expression patterns from IAA9 during wood
formation (Fig. S2). Aux/IAAs usually require functional coopera-
tion of ARFs via protein interactions, and harbor variable interact-
ing affinities with different ARFs (Vanneste & Friml, 2009).
Thus, specific ARF partners of PttIAA3 and PtoIAA9 may cause
their differential regulation on cambial activity.
Compared to WT, the proportion of woody tissues of transgenic
plants overexpressing PtoIAA9m was significantly reduced (Fig. 2e,
f), implying inhibition of secondary xylem development by
PtoIAA9. Both reduced xylem cell layers and restricted xylem cell
size led to PtoIAA9-inhibited wood formation that was rescued by
overexpressing PtoARF5 (Figs2,4,5).Cambialproliferationand
cambium specification to xylem cooperatively determine the num-
ber of xylem cell layers (Sanchez et al., 2012). Given the excluded
PtoIAA9-mediated effects on cambial proliferation, we propose that
the reduced periclinal cell division rate may be responsible for
PtoIAA9-repressed wood formation. This is supported by the disap-
pearance and reappearance of the EDX cell layers in PtoIAA9-
overexpressing and PtoARF5.1-complementing lines, respectively
(Figs 2, 5). These cell layers are initially specified from cambial cells
and at early stage of wood formation. Active cambium periclinal cell
division leads to rapid xylem mother cell accumulation, while a low
rate of cambium periclinal cell division slowly produces xylem
mother cells, resulting in an absence of the EDX cell layers in the
PtoIAA9m-OE lines. Similar phenotypes were also found in trees
during dormancy (Gricar et al., 2014). Therefore, specification and
expansion of xylem cells during wood differentiation are coordi-
nated by IAA9-ARF5-mediated auxin signaling in poplar.
Recent studies have shown that Arabidopsis MP/ARF5 directly
represses WOX4 expression for restrained stem cell quantity
(Brackmann et al., 2018), and switches of MP/ARF5 phosphory-
lation mediated by BIL1 kinase integrate peptide and cytokinin
signaling for cambial activity (Han et al., 2018). Here we provide
evidence in Populus that ARF5 coordinates multiple behaviors for
woody cell differentiation via directly targeting HB7/8 paralogs.
BDL/IAA12 is a canonical Aux/IAA partner of ARF5/MP in Ara-
bidopsis during embryogenesis (Hamann et al., 2002). Due to
promiscuous protein interactions between Aux/IAAs and ARFs,
cooperative functions of IAA12-ARF5 could not be excluded in
poplar. The significantly lower transcript abundance of IAA12
paralogs than IAA9 and their different expression patterns in
wood-forming stems (Fig. S2) suggest possible variable roles in
wood differentiation. We expected that IAA9 also regulates pro-
cambial specification during root development and leaf vein pat-
tering. Indeed, the knockdown of IAA9 in tomato led to
abnormal leaf formation with different vascular tissue patterning
(Wang et al., 2005).
Arabidopsis HD-ZIP III transcription factors are key regula-
tors in orchestrating vascular patterning in developmental
contexts of root, leaf and stem (Ramachandran et al., 2017).
AtHB8 displays specific expression in procambial cells and is
involved in vascular differentiation (Baima et al., 2001). HB7,
the poplar ortholog of AtHB8, has been revealed to be a dose-
dependent regulator that balances cambium activity and xylem
differentiation during secondary growth (Zhu et al., 2013). We
found that a series of core and canonical auxin response elements
(AuxREs), the potential DNA binding sites for ARFs, are present
in the promoters of poplar HB7 and HB8. ChIP and effector–re-
porter tests revealed transcriptional activation of PtoARF5.1 via
Fig 8 A model for Aux/IAA-ARF-HB-dependent wood formation in
Populus tomentosa. Auxin displays complex regulation of various steps
during wood formation in poplar. The auxin-dependent cambial cell
division was demonstrated by Nilsson et al. (2008). The role of auxin in
xylem maturation, mainly including secondary wall deposition, remained
unclear. In this study, PtoIAA9-ARF5-HB7/8-mediated wood
differentiation is proposed. The auxin-dependent PtoIAA9-ARF5 module
directly regulates the expression of PtoHB7/8, which drives multiple
cellular events including xylem cell specification, expansion and vessel
formation during secondary xylem development. Arrows represent a
positive regulatory action of one component on another. Lines ending
with a trait represent a negative regulatory action. Dotted lines indicate
indirect regulation.
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direct binding to the AuxRE elements harbored in the promoters
of HB genes (Fig. 6). Importantly, PtoHB7 is able to functionally
rescue phenotypes of xylem cell specification, expansion and ves-
sel density (Fig. 7). Therefore, our data suggested that the
PtoIAA9–PtoARF5 module directly targets PtoHB7 and PtoHB8
for regulating secondary cell differentiation. In Arabidopsis,
ARF5/MP has been identified to control leaf vein patterning by
directly targeting AtHB8 (Donner et al., 2009), demonstrating
ARF5-HB7/8 as a conserved pathway for vascular patterning of
leaves and stems in herbaceous and woody species. Moreover, the
HB8 orthologs can directly regulate the expression of ACL5
encoding thermospermine synthase in both Arabidopsis and
poplar for xylem differentiation, and also form a feedback regula-
tion on the auxin pathway (Milhinhos et al., 2013; Baima et al.,
2014). We also found that the expression of ACL5 was signifi-
cantly decreased by PtoIAA9 overexpression and rescued by
PtoARF5 complementation (Fig. S11).
Collectively, our data allow us to propose a model for
IAA9-ARF5-mediated coordination for wood differentiation
(Fig. 8). Auxin releases the repression of PtoIAA9, by trigger-
ing its protein degradation, on PtoARF5-activated auxin
responsive gene expression during cambium-derived wood
formation. In parallel, auxin-inducible transcript abundance
of PtoIAA9 switches-off auxin signaling in a self-controlled
manner. The PtoIAA9–PtoARF5 module directly targets
PtoHB7 and PtoHB8 to mediate auxin regulation on multi-
ple cellular events, including xylem specification, expansion
and vessel formation for secondary xylem development. In
conclusion, our findings show that the auxin-Aux/IAA-ARF
pathway plays a key role in controlling wood formation via
HB-driven cambium differentiation in Populus.
Acknowledgements
We thank Drs Guoqing Niu, Xinqiang He and Jianquan Liu for
helpful comments. This work was supported by the National
Natural Science Foundation of China (31500544, 31670669,
31870657, 31800505 and 31870175), National Key Project for
Research on Transgenic Plant (2016ZX08010-003) and Funda-
mental Research Funds for the Central Universities
(XDJK2018AA005, XDJK2014a005). The authors declare no
conflicts of interest.
Author contributions
KL, CX and HCW designed the work; CX, FH, YS, XF, WL,
CL, HY, YL and DF performed experiments and data analyses;
CX drafted the manuscript; KL and HCW revised the
manuscript. All authors approved the final version of the
manuscript for publication. CX, YS, FH, XF and HY contributed
equally to this work.
ORCID
Keming Luo https://orcid.org/0000-0003-4928-7578
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Supporting Information
Additional Supporting Information may be found online in the
Supporting Information section at the end of the article.
Dataset S1 Differentially expressed genes in the RNAseq-based
transcriptomic analysis of PtoIAA9m-OE vs WT.
Fig. S1 Phylogenetic relationship of poplar and Arabidopsis Aux/
IAA family members.
Fig. S2 Expression levels and patterns of poplar Aux/IAA genes in
wood-forming stem tissues.
Fig. S3 Sequence alignment of PtoIAA9, Arabidopsis IAA8/9
and tomato IAA9.
Fig. S4 Attributes of a typical Aux/IAA protein harbored by the
IAA9 protein.
Fig. S5 Generation and stem phenotypes of PtoIAA9m-OE
poplar lines.
Fig. S6 Sequence, expression and protein interactions of ARF5s
in poplar.
Fig. S7 Constitutive expression of PtoARF5 in the PtoIAA9m-OE
lines.
Fig. S8 Comparative transcriptomic analysis of the PtoIAA9m-
OE and WT lines via RNAseq.
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Fig. S9 Regulation of the PtoIAA9/ARF5 module on PtoHB7/8
promoter activities via effector–reporter tests using GUS as a
reporter gene.
Fig. S10 Constitutive expression of PtoHB7 in the PtoIAA9m-
OE lines.
Fig. S11 Expression of some secondary xylem development-
related genes in PtoIAA9m-OE, PtoARF5.1- and PtoHB7-com-
plementing transgenic lines.
Methods S1 Experimental methods for Figures S4 and S8,
including subcellular localization, protein stability assay and
mRNA sequencing.
Table S1 Primer sequences used in this study.
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