\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[ngerman,polish,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Liana Attachment to Supports Leads to Profound Changes in Xylem Anatomy
and Cambium and Differentiating Xylem Transcriptional Profile}



\author[1]{André Carvalho Lima}%
\author[1]{Sónia Cristina da Silva Andrade}%
\author[1]{Caian Souza Gerolamo}%
\author[1]{Diego Souza}%
\author[2]{Luiz Lehmann Coutinho}%
\author[1]{Magdalena Rossi}%
\author[1]{Veronica Angyalossy}%
\affil[1]{Universidade de Sao Paulo Instituto de Biociencias}%
\affil[2]{Universidade de Sao Paulo Escola Superior de Agricultura Luiz de Queiroz}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Wood serves crucial functions in plants, yet our understanding of the
molecular regulation governing the composition, arrangement, and
dimensions of its cells remains limited. The abrupt change in wood
anatomy of lianas represents an excellent model to address the
underlying mechanism, although consistent triggering factors for this
process remain uncertain. In this study we examined how physical support
attachment impacts the development of lianescent xylem anatomy in
\emph{Bignonia magnifica} (Bignoniaceae), employing a comprehensive
approach integrating detailed anatomical analysis with gene expression
profiling of cambium and differentiating xylem. Our findings demonstrate
that attachment to physical supports triggers the formation of
lianescent xylem, leading to increased vessel size, range of vessel
sizes, broader vessel distribution, reduced fiber content, and higher
potential specific water conductivity. These shifts in wood anatomy
coincide with the downregulation of genes associated with cell division
and cell wall biosynthesis, and the upregulation of transcription
factors (TFs), defense/cell death, and hormone-responsive genes in the
lianescent xylem. Based on our results, we propose a model delineating
the molecular control underlying the formation of lianescent xylem,
revealing how the increased complexity of lianescent anatomy reflects a
more intricate transcriptional regulatory network encompassing a more
diverse repertoire of TFs and hormone-responsive genes.%
\end{abstract}%



\sloppy


\textbf{Liana Attachment to Supports Leads to Profound Changes in Xylem
Anatomy and Cambium and Differentiating Xylem Transcriptional Profile}

Andr\selectlanguage{ngerman}é Carvalho Lima\textsuperscript{1,2,*}, Sónia Cristina da Silva
Andrade\textsuperscript{3}, Caian Souza Gerolamo\textsuperscript{1},
Diego Trindade de Souza\textsuperscript{1}, Luiz Lehmann
Coutinho\textsuperscript{4}, Maria Magdalena Rossi\textsuperscript{1},
Veronica Angyalossy\textsuperscript{1}

\textsuperscript{1} Departamento de Botânica, Instituto de Biociências,
Universidade de São Paulo, São Paulo, Brazil. \textsuperscript{2}Present
address: Faculdade de Ciências Agronômicas, Universidade Estadual
Paulista, Botucatu, Brazil. \textsuperscript{3} Departamento de Genética
e Biologia Evolutiva, Instituto de Biociências, Universidade de São
Paulo, São Paulo, Brazil. \textsuperscript{4} Departamento de Zootecnia,
Escola Superior de Agricultura `Luiz de Queiroz' (ESALQ), Universidade
de São Paulo, Piracicaba, Brazil.

*Corresponding author: André C Lima - andrec.lima@gmail.com

\textbf{ORCIDs}

André C Lima: 0000-0001-9183-3064

Sónia CS Andrade: 0000-0002-1302-5261

Caian S Gerolamo: 0000-0003-1819-5371

Diego T de Souza: 0000-0001-8783-2333

Luiz L Countinho: 0000-0002-7266-8881

Magdalena Rossi: 0000-0003-3650-772X

Veronica Angyalossy:0000-0002-9823-9946

\textbf{Summary}

Wood serves crucial functions in plants, yet our understanding of the
molecular regulation governing the composition, arrangement, and
dimensions of its cells remains limited. The abrupt change in wood
anatomy of lianas represents an excellent model to address the
underlying mechanism, although consistent triggering factors for this
process remain uncertain. In this study we examined how physical support
attachment impacts the development of lianescent xylem anatomy in
\emph{Bignonia magnifica} (Bignoniaceae), employing a comprehensive
approach integrating detailed anatomical analysis with gene expression
profiling of cambium and differentiating xylem. Our findings demonstrate
that attachment to physical supports triggers the formation of
lianescent xylem, leading to increased vessel size, range of vessel
sizes, broader vessel distribution, reduced fiber content, and higher
potential specific water conductivity. These shifts in wood anatomy
coincide with the downregulation of genes associated with cell division
and cell wall biosynthesis, and the upregulation of transcription
factors (TFs), defense/cell death, and hormone-responsive genes in the
lianescent xylem. Based on our results, we propose a model delineating
the molecular control underlying the formation of lianescent xylem,
revealing how the increased complexity of lianescent anatomy reflects a
more intricate transcriptional regulatory network encompassing a more
diverse repertoire of TFs and hormone-responsive genes.

\textbf{Keywords:} gene expression profile; lianas; transcriptome; wood;
wood traits; secondary xylem; differentiation.

\textbf{Introduction}

Wood serves three essential functions in plants: providing mechanical
support, positioning leaves and reproductive organs for photosynthesis
and pollination/dispersion; conducting water and nutrients; and storing
water and nutrients (Baas \emph{et al.} , 2004). The evolution of
cellular specialization in Angiosperm wood, or secondary xylem, has led
to a division of labor among different cell types. Fibers primarily
provide mechanical support, vessels conduct water and minerals, and
axial and radial parenchyma store resources (Tyree \& Zimmermann, 2002).
Secondary xylem formation occurs through the activity of the cambium, a
lateral meristem, producing the different cell types of the secondary
xylem toward the inside of the stem, and secondary phloem toward the
exterior (Evert, 2006).

Studies on model species, like \emph{Arabidopsis thaliana}
and\emph{Populus} have significantly advanced the understanding of
cambium function and activity, and xylem differentiation (Groover, 2005;
Robischon \emph{et al.} , 2011; Ye \& Zhong, 2015). Crucial molecular
components, such as the \emph{TDIF/CLE41/CLE44-TDR/PXY-WOX4} module
controlling cambial maintenance and proliferation (Hirakawa \emph{et
al.} , 2010; Zhang \emph{et al.} , 2019), and the secondary cell wall
(SCW) biosynthesis master transcription factors NACs (\emph{NST1, XND1,
VND6, VND7,} and \emph{SND1} ) and MYBs (Kubo \emph{et al} ., 2005;
Zhong\emph{et al} ., 2006, 2007; Zhang \emph{et al.} , 2020), have been
characterized in \emph{A. thaliana.}These findings have proven to be
conserved among diverse herbaceous and woody species (Hu \emph{et al} .,
2010; Zhong \emph{et al} ., 2011; Hirakawa \& Bowman, 2015). However,
our understanding of the molecular regulation underlying the cellular
composition, arrangement, and dimensions of secondary xylem, crucial for
wood functions, remains limited (Ziemi\selectlanguage{polish}ń\selectlanguage{english}ska \emph{et al.} , 2015;
Beeckman, 2016).

Advancements in technologies, like high-throughput sequencing have
enabled molecular studies in non-model species, revealing unique
features and processes absent in model species organisms
(Carpentier\emph{et al.} , 2008; Wang \emph{et al} ., 2009). Within this
context, lianas exhibit a distinctive set of wood anatomical
characteristics associated with high flexibility, conduction efficiency,
and intraindividual plasticity in wood traits (Fig. 1). These attributes
make lianas an excellent model for investigating vascular system
differentiation. Despite evolving independently in various plant groups,
most lianas share convergent secondary xylem anatomical features,
including reduced fibers, wide vessels (up to 500 \selectlanguage{greek}μ\selectlanguage{english}m in diameter)
associated with small vessels (referred to as vessel dimorphism by
Carlquist, 1981), and the presence of soft tissues interspersed in the
xylem (Schenck, 1893; Obaton, 1960; Carlquist, 1985). These common
features are collectively known as the ``lianescent vascular syndrome''
(Angyalossy \emph{et al.} , 2015).

Most lianas, however, show a dense fibrous xylem with small vessels,
resembling the xylem of self-supporting species at the beginning of
secondary development (Schenck, 1893; Obaton, 1960; Caball\selectlanguage{ngerman}é, 1998). The
transition from the formation of this dense xylem (termed as
self-supporting xylem hereafter) to the development of that showing the
lianescent vascular syndrome (termed as lianescent xylem hereafter)
occurs abruptly, as seen in adult stem cross-sections (Fig. 1b).
Importantly, self-supporting and lianescent xylems are produced
simultaneously along liana stems. While older sections produce
lianescent xylem, younger parts continue to form self-supporting xylem.
Furthermore, recent findings indicate that attachment to supports
triggers the production of lianescent xylem in the twining liana
\emph{Condylocarpon guianensis}(Soffiatti \emph{et al} ., 2022).
However, it remains unclear if this triggering factor is consistent
across lianas from different lineages or with different climbing
methods.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Fig. 1.} \emph{B. magnifica} wood plasticity seen in stem
cross-sections along the stem. (\textbf{a} ) A one-year-old plant grown
with support. (\textbf{b} ) Secondary xylem formed by the regular
cambium (RC) in older portions of the stem shows an abrupt transition
from the self-supporting xylem (Ss) formed at the beginning of
development to the lianescent xylem (L). The lianescent xylem is
characterized by the presence of wide vessels, which are associated with
narrow vessels and fewer fibers. (\textbf{c} ) The secondary xylem in
younger portions of the stem is composed only of the self-supporting
xylem (SS) formed by a high proportion of fibers and small vessels. It
is possible to see the innermost position of the four regions of variant
cambium (VC), responsible for the formation of phloem wedges
characteristic of Bignonieae lianas, intercalated with the regular
cambium (RC). Scales: (a) 33 cm, (b) 1 mm, (c), 500 \selectlanguage{greek}µ\selectlanguage{english}m. P: pith; Ph:
secondary phloem.

Here we tested the hypothesis that liana attachment to physical supports
promotes the formation of lianescent xylem by the cambium in older stem
parts in the tendril climber \emph{Bignonia magnifica} (Bignoniaceae).
We propose that intraindividual plasticity in wood anatomy results from
remodeling of the cambium and differentiating xylem transcriptome in
these regions. By characterizing the anatomy of self-supporting and
lianescent xylem, analyzing differential gene expression in the cambium
and differentiating xylem, and providing a manually-curated
comprehensive annotation, we propose a model for the molecular control
of lianescent xylem differentiation. Our findings indicate distinct
expression patterns between self-supporting and lianescent phases, with
upregulation of cell division and cell wall-related transcripts in
self-supporting xylem and a more intricate transcriptional regulation
network, involving a diverse repertoire of transcription factors (TFs)
and hormone-responsive genes.

\textbf{Material and Methods}

\textbf{Plant Material}

\emph{Bignonia magnifica} W. Bull, Bignoniaceae, is native to Central
America and northern South America (Lohmann \& Taylor, 2014). It belongs
to the monophyletic tribe Bignonieae, which is predominantly composed of
lianas that climb using terminal leaflet-modified tendrils. Bignonieae
is the most diverse and abundant clade of lianas in neotropical lowland
forests (Gentry, 1986). A synapomorphy of the tribe is the presence of
four or multiples of four, equidistant phloem wedges formed by variant
regions of the cambium, which are located more internally in the stem
due to its reduced xylem production (Lohmann \& Taylor, 2014;
Pace\emph{et al.} , 2015; Fig. 1c).

\textbf{Growth Conditions}

Clones of \emph{Bignonia magnifica} were obtained from cuttings of a
single plant cultivated at the gardens of the Biosciences Institute of
the University of S\selectlanguage{ngerman}ão Paulo, Brazil (23.56\textsuperscript{o} S,
46.73\textsuperscript{o} W). The average annual temperature in the
region is 21.5\textsuperscript{o}C and the mean total annual
precipitation is approximately 1600 mm (Brazil National Weather
Institute -- www.inmet.gov.br).

Sixteen one-year-old plants, each measuring one meter in height, were
transplanted to pots measuring 60 cm in diameter and 50 cm in height
with soil rich in organic matter in April 2016 (Fig. 2a). Six of these
plants were grown with physical supports (stainless-steel wire meshes,
2.1 mm diameter wire, 7x7 cm mesh, Fig. 2b) for two years: five plants
were used for self-supporting and lianescent secondary xylem anatomy
analysis, and one plant was selected for the collection of cambium and
differentiating xylem samples for transcriptome assembling and
differential gene expression analysis. The remaining ten plants had
their lateral branches and main stem apices pruned to stimulate the
production of new branches of the same age (Fig. 2c). These ten plants
were divided into two groups for the analysis of physical support impact
on the lianescent xylem formation: one group was grown with physical
supports (Fig. 2d), and the other group was grown without support aid
(Fig. 2e). Pots were spaced 1.5 m apart and were watered to a maximum
field capacity three times a week for the first year and weekly
thereafter.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 2.} Schematic diagram showing plant material and growth
conditions. (a) Plant material consisted of 16 clones propagated through
cuttings. (b) Six plants were grown besides physical supports (wire
meshes) for two years: five plants were used for self-supporting and
lianescent xylem (X.) anatomy characterization, carried at 4/5 of the
distance from the shoot apex to the base, in the same cross-section; and
one plant had the cambium and differentiating xylem sampled for
transcriptome assembling and differential gene expression analysis.
Tissue was sampled from 60 cm from the shoot apex (around the
10\textsuperscript{th} internode) to the base of the stem
(\emph{ca.}20\textsuperscript{th} internode). (c) Ten plants had lateral
branches and main stem apices pruned to stimulate the production of new
branches of the same age. They were grown for 32 months and were used to
assess the relationship between lianescent xylem production and physical
support attachment. (d) Five pruned plants were grown beside physical
supports. (e) The other five pruned plants were grown without physical
support. Scale bars: (a) and (c) 25 cm; (b), (d) and (e) 150 cm.

\textbf{Xylem Anatomy Analysis}

To characterize self-supporting and lianescent xylem anatomy, we sampled
the longest stems from the five unpruned plants (Fig. 2b after two years
of cultivation, in May 2018. Due to differences in sampled stem lengths,
which ranged from 3.6 to 8.4 m, self-supporting and lianescent xylems
were characterized at 4/5 of the distance to the base, in the same
cross-section. After collection, the samples were immediately fixed in
FAA 50 (10\% formalin, 5\% acetic acid, 50\% alcohol). Subsequently, the
samples were rehydrated, embedded with polyethylene glycol 1500, and
transversal and longitudinal sections from 8 to 15 \selectlanguage{greek}µ\selectlanguage{english}m in thickness were
made using a sliding microtome, as described by Barbosa \emph{et
al.}(2010). Additionally, portions of self-supporting and lianescent
xylem were dissected under a stereomicroscope and macerated in
Franklin's solution (Berlyn \& Miksche, 1976).

Photomicrographs were captured using a Leica DML photomicroscope
attached to a digital camera DFC 310FX, and measurements were performed
using ImageJ v1.52a (Schneider \emph{et al.} , 2012). Secondary xylem
characterization was based on the IAWA List of Microscopic Features for
Hardwood Identification (IAWA Committee, 1989) and Scholz \emph{et al.}
(2013). Secondary xylem characters were analyzed with a magnification of
200\selectlanguage{ngerman}× or 400×, while the intervessel pit diameter was the only analyzed
parameter requiring a higher magnification of 1,000× under oil
immersion. Relative areas of fibers, axial parenchyma, and rays were
calculated by analyzing six 0.1 mm² images from random portions of the
transverse section (Gerolamo \& Angyalossy, 2017). The amount of xylem
produced by the cambium was measured by calculating the average length
of three straight lines from the cambium to the pith. The vessel
grouping index was measured by the ratio of total number of vessels to
total number of vessel groupings, including solitary and grouped vessels
(Scholz et al. 2013). The equivalent vessel diameter
(D\textsubscript{v;} \selectlanguage{greek}µ\selectlanguage{english}m) was calculated by measuring the vessel lumen
area (A; \selectlanguage{greek}µ\selectlanguage{english}m\textsuperscript{2}) and using the formula:

\emph{D\textsubscript{v}} =
(4\emph{A}\(\pi\)\textsuperscript{-1})\textsuperscript{1/2}

The hydraulic diameter (D\textsubscript{h}), corresponding to the mean
diameter that all sampled vessels would have to correspond to the total
conductivity of the same number of vessels (n) (Tyree \emph{et al.} ,
1994, Scholz \emph{et al.} , 2013) was calculated using the formula:

\begin{equation}
D_{h}=(\Sigma_{1}^{n}\ D_{v}^{4}\ n^{-1})^{1/4}\nonumber \\
 \end{equation}

Potential specific conductivity (K\textsubscript{p}; kg
m\textsuperscript{-1} Mpa\textsuperscript{-1} s\textsuperscript{-1}) was
calculated following Poorter \emph{et al.} (2010) using the formula:

K\textsubscript{p} = (\(\pi\)
\selectlanguage{greek}ρ\selectlanguage{english}\textsubscript{w}128\selectlanguage{greek}η\selectlanguage{english}\textsuperscript{-1})
V\textsubscript{d}\emph{D\textsubscript{v}} \textsuperscript{4}

where D\textsubscript{h} (m) is the vessel hydraulic diameter,
\selectlanguage{greek}ρ\selectlanguage{english}\textsubscript{w} is water density at 20 \selectlanguage{ngerman}°C (998.2
kg.m\textsuperscript{-3}), \selectlanguage{greek}η \selectlanguage{english}is the viscosity index of water (1.002 x
10\textsuperscript{-9} MPa s\textsuperscript{-1} at 20\selectlanguage{ngerman}°C), and
V\textsubscript{d} is vessel density (m\textsuperscript{-2}). Sample
sizes were recommended by Scholz \emph{et al.} (2013). Data normality
and variance homogeneity were tested using Shapiro-Wilk and Levene's
tests, respectively. To compare each self-supporting and lianescent
xylem anatomy characteristics, the Student's t-test for parametric data,
and the Mann-Whitney test for nonparametric data were applied in R
software v.3.6.1 (R Core Team, 2014).

\textbf{Effect of Support on Lianescent Xylem Formation}

To investigate the impact of support availability on the formation of
lianescent xylem, we analyzed plants cultivated for 32 months, both with
and without support (Fig. 2d and 2e, respectively).
Two-centimeter-length stem samples were collected at regular 50 cm
intervals, starting from the apex and avoiding nodes and potential
injury. Subsequently, fresh cross-sections 10 to 25 \selectlanguage{greek}µ\selectlanguage{english}m in thickness were
obtained using a sliding microtome. Sections were then mounted unstained
on 50\% glycerin semi-permanent slides, or double stained with 1\% Astra
Blue and 1\% Safranin (Bukatsch, 1972). Photomicrographs were captured
as previously described.

\textbf{Samples for Differential Expression Analysis}

To comprehend the transcriptional control mechanism responsible for the
concomitant formation of the self-supporting and lianescent xylem in
different portions of the same plant of \emph{B. magnifica} , we sampled
the cambium and differentiating xylem of entire internodes samples
(approximately 30 cm long) collected from 60 cm from the shoot apex to
the base of the stem (from around the 10\textsuperscript{th} to the
20\textsuperscript{th} internodes), along different stems of a single
plant. After two years of cultivation, the sampling was conducted in
April 2018 during the period of active cambium by peeling the bark and
gently scraping the exposed xylem (Allona \emph{et al.} , 1998; Sterky
\emph{et al.} , 1998; Perry \emph{et al.} , 2021). The cambial activity
was assessed by anatomical analysis (Supporting Information Fig. S1) and
the ease with which the bark was peeled (Wilcox, 1962; Melder \emph{et
al.} , 2015). We chose this sampling strategy to avoid that the observed
differences could be a product of, or be masked by, allelic variations,
as \emph{B. magnifica} is a wild, highly heterozygous species with no
available sequence data. Given our focus on cambium and differentiating
xylem, we also avoided sampling tissues below the peeled bark to prevent
collecting other tissues, such as phloem and cortex, as the bark in the
species was fragile and wrapped immediately after removal. This also
prevented sampling the cambium and phloem from the phloem wedges
(Supporting Information Fig. S2), what would add complexity to our
analysis and was out of the scope of the present work. The innermost
position of the variant cambium in the stem facilitated the collection
of only regular portions for differential expression analysis, which
were the only ones analyzed for this purpose. Tissues were immediately
frozen in liquid nitrogen and stored at -80 \selectlanguage{ngerman}°C. Before tissue harvest,
each stem segment had its base cut and stored in 50 \% alcohol to
determine the xylem phase, self-supporting or lianescent, and any
segment showing dubious or transitional anatomy, and the segment next to
it were discarded. Six pools of self-supporting and six pools of
lianescent cambium and differentiating xylem, composed of 8-19 segments
each, were collected for sequencing and differential expression
analysis.

\textbf{RNA Extraction, Library Construction, and Sequencing}

We extracted total RNA from the collected samples after sample
lyophilization using ReliaPrepTM RNA Tissue Miniprep System (Promega
Corporation) following manufacturer protocol. The quantity and quality
of the extracted RNA were assessed using a Qubit (Thermo Scientific),
while RNA integrity was confirmed by 1 \% (w/v) agarose gel
electrophoresis and by a Bioanalyzer 2100 (Agilent Technologies) using
the RNA 6000 Nano LabChip Kit. RNA samples with RIN \selectlanguage{english}[?] 6 were considered
suitable for sequencing.

Libraries for RNA sequencing were constructed using the TruSeq Stranded
mRNA Sample Prep LT Protocol (Illumina), and were sequenced with 2x100
bp paired-end reads on an Illumina HiSeq2500 platform, using a HiSeq
Flow Cell v4 with HiSeq SBS Kit v4 (Illumina) at the Centro de Genomica
Funcional Aplicada a Agropecuaria e Agroenergia, ESALQ, USP, Brazil.

\textbf{Sequence Analysis}

Adaptors and low-quality sequences below 23 and 30 Phred quality
parameters for maximum average error and maximum error at the end,
respectively, were trimmed using SeqyClean v.1.9.10 (Zhbannikov \emph{et
al} ., 2017). Only high-quality paired-end sequences were used for
further analysis. Contaminant sequences were removed using HISAT2 v2.0.5
(Kim \emph{et al} ., 2015). For contaminant identification, a
contaminant bank was built containing sequences of Bacteria, Nematoda,
Oomycetes, and Platyhelminthes with the assembled complete genome,
chromosome, or scaffold from reference or representative genomes (RefSeq
category). Data were downloaded in December 2017 using NCBI Entrez
Direct E-utilities v6.60 (Kans, 2018). Quality-filtered reads were
aligned against the contaminant bank database using the HISAT2 flag
''un-conc'' to recover paired-end sequences that failed to align
concordantly with contaminants. \emph{De novo} transcriptome assembling
was generated using Trinity v.2.8.3 (Haas \emph{et al} ., 2013) with
default parameters. The longest isoforms were extracted using the
TRINITY package script
get\_longest\_isoform\_seq\_per\_trinity\_gene.pl.

The completeness of our transcriptome assembly was assessed using the
Benchmarking Universal Single-Copy Ortholog v5.2.2 (BUSCO,
http://busco.ezlab.org, Manni\emph{et al.} , 2021). The\emph{de novo}
assembled transcriptome was annotated against the Viridiplantae
SwissProt database (www.uniprot.org) (downloaded on September 18, 2019)
using the BLASTX program, of BLAST suite (Camacho\emph{et al.} , 2008),
with 1e\textsuperscript{-5} e-value threshold. Annotated assemblies had
their GeneOntology (GO) terms retrieved with the tool ''Retrieve/ID
mapping'' on the UniProt website (www.uniprot.org), and parental terms
were recovered using Blast2GO (version 5.2.5) ''Combined Graph'' tool
(Conesa \emph{et al} ., 2005).

Further, functional annotation of \emph{B. magnifica} cambium and
differentiating xylem transcriptome was compared to that of the model
species \emph{Populus} (\emph{P. x euramericana} ) and \emph{Eucalyptus
grandis} annotated by Zinkgraf \emph{et al} .(2017) over the data
generated by Xu \emph{et al.} (2014). \emph{A. thaliana} orthologs of
each expressed gene from the two model species retrieved by the authors
were used to recover the associated GO terms using the UniProt website.
For transcriptome comparison, we selected the Biological Process GOs
associated with at least 10 \% of the transcripts using the Blast2GO
''Combined Graph'' tool for all three species. A heat map created to
visualize the functional comparison between \emph{B. magnifica}
,\emph{Populus,} and \emph{Eucalyptus} was produced using the online
tool Morpheus (Broad Institute, URL:
https://software.broadinstitute.org/morpheus/).

For differential expression analysis, the 12 RNA-Seq samples were mapped
over the GO term-associated transcripts using Salmon v.0.11.3 (Patro
\emph{et al} ., 2017). The transcript abundances were then used to
identify the differentially expressed genes (DEGs) between
self-supporting and lianescent-phases. The significance of differences
was assessed with the package edgeR v.3.26.8 (Robinson \emph{et al} .,
2010) from Bioconductor v.3.9 software (Huber \emph{et al} ., 2015). For
this purpose, we normalized the 12 libraries with the TMM method
(Robinson \& Oshlack, 2010) using the calcNormFactors() function, while
common dispersions were calculated with the estimateCommonDisp()
function (Robinson \& Smyth, 2008). The threshold adopted for
significance assessing was p \textless{} 0.05, false discovery rate
(FDR) \textless{} 0.05, and log2 fold change \textgreater{} 2. GO
functional enrichment analysis of DEGs was performed by Fisher exact
test (FDR \textless{} 0.05) using Blast2GO (version 5.2.5).

\textbf{Results}

\textbf{Vessel Size, Distribution, and Fiber Relative Area Differentiate
Self-Supporting and Lianescent Xylem}

To characterize the phenotypic changes in \emph{B. magnifica} wood, we
examined the xylem anatomy of mature stems showing the self-supporting
and lianescent xylem. The careful analysis of multiple images of each
sectional plane showed that the self-supporting xylem consisted of a
matrix of septate fibers and rays, with vessels and scanty paratracheal
axial parenchyma produced solely by the fascicular cambium, opposite the
primary xylem poles (Fig. 3a, left). This restriction on vessel
production was sustained throughout the self-supporting phase,
conferring a radial arrangement to the vessels opposite the protoxylem
poles. The rays in the self-supporting xylem were uniseriate and
composed of upright and square cells (Fig. 3b, left).

In contrast, the onset of the lianescent xylem was marked by the
simultaneous production of several large vessels throughout the cambium
width by both the fascicular and interfascicular cambium (Fig. 3a, white
arrows). These large vessels had a diameter of 111\selectlanguage{greek}µ\selectlanguage{english}m and were solitary
or associated with small vessels and formed a bimodal distribution of
vessel diameter classes (Fig. 3c). The lianescent xylem also had septate
fibers, but the rays were uni to biseriate and composed of predominantly
procumbent and square cells (Fig. 3b, right).

Quantitative analysis revealed significant differences between the two
phases (Table 1). Vessel relative area increased from 5.7\% in the
self-supporting phase to 35.9\% in the lianescent xylem. This increase
was mainly at the expense of fiber relative area, which decreased from
81.6\% in self-supporting xylem to 54.7\% in lianescent xylem. The
increase in vessel relative area was primarily driven by vessel size,
while vessel density remained constant. Consequently, the potential
specific conductivity of the xylem increased more than 20-fold, from
4.52 \selectlanguage{ngerman}× 10\textsuperscript{-6} kg
m\textsuperscript{-1}Mpa\textsuperscript{-1} s\textsuperscript{-1} in
self-supporting xylem to 106.98 × 10\textsuperscript{-6} kg
m\textsuperscript{-1}Mpa\textsuperscript{-1} s\textsuperscript{-1} in
lianescent xylem. Intervessel-pit diameter and vessel grouping index,
both related to water conduction, also increased, while fibers in the
lianescent xylem were shorter, wider, and had thicker cell walls
compared to the self-supporting xylem. These changes in cell dimensions
resulted in an overall reduction in cell density (number of cells per
area).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\par\null

\textbf{Fig. 3.} Self-supporting and lianescent xylem anatomy.
(\textbf{a} ) Stem cross-section showing self-supporting xylem
(\textbf{SS)} , characterized by small radially arranged vessels formed
only by the fascicular cambium, opposite to protoxylem poles (black
arrows), and a high proportion of fibers. The beginning of the
lianescent xylem (L) is marked by the concomitant production of several
large vessels (white arrows) not restricted to radial files and the
reduction of fiber amount. The large vessels are associated with small
vessels and comprise a larger proportion of the xylem in this phase.
(\textbf{b} ) Longitudinal radial section of the xylem. Ray composition
changes from upright cells in \textbf{SS} to mainly square and
procumbent cells in \textbf{L} (white arrows). Septate fibers
(arrowheads) are found in both phases. Vessel (v). (\textbf{c} )
Self-supporting and lianescent xylem vessel diameter classes
distribution. Scale bars: (a) 125 \selectlanguage{greek}μ\selectlanguage{english}m; (b) 100 \selectlanguage{greek}μ\selectlanguage{english}m.

This analysis highlights the distinct characteristics of self-supporting
and lianescent xylem, including the broader distribution of vessels in
the later phase, no longer restricted to the fascicular regions. The
production of large vessels in the lianescent phase significantly
increased water potential specific conductivity and reduced the number
of cells per area.

\textbf{Table 1.} Quantitative characterization of self-supporting and
lianescent xylem.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Relative areas of different cell types (\%)} &
\textbf{Self-supporting} & \textbf{Lianescent} & \textbf{Difference
between phases (\%)}\tabularnewline
Vessels & \selectlanguage{ngerman}\textbf{5.7 (±2.7)} & \selectlanguage{ngerman}\textbf{35.9 (±8.5)} & \textbf{529.82
**}\tabularnewline
Fibers & \selectlanguage{ngerman}\textbf{81.6 (±3.8)} & \selectlanguage{ngerman}\textbf{54.7 (±8.5)} & \textbf{-32.97
**}\tabularnewline
Axial parenchyma & \selectlanguage{ngerman}\textbf{0.6 (±0.4)} & \selectlanguage{ngerman}\textbf{1.7 (±0.9)} &
\textbf{183.33 *}\tabularnewline
Radial parenchyma & \selectlanguage{ngerman}\textbf{12.0 (±2.5)} & \selectlanguage{ngerman}\textbf{7.7 (±1.6)} &
\textbf{-35.83 *}\tabularnewline
\textbf{Vessels} & & &\tabularnewline
Mean diameter (D - \selectlanguage{greek}µ\selectlanguage{english}m) & \textbf{26.29} (\selectlanguage{ngerman}\textbf{±}16.59 & \selectlanguage{ngerman}\textbf{45.57
(±37.96)} & \textbf{73.34 ***}\tabularnewline
Max diameter (D\textsubscript{max} - \selectlanguage{greek}µ\selectlanguage{english}m) & \textbf{73.48}
(\selectlanguage{ngerman}\textbf{±}12.25) & \textbf{141.20} (\selectlanguage{ngerman}\textbf{±}10.96) & \textbf{92.16
***}\tabularnewline
Minimum diameter (D\textsubscript{min} - \selectlanguage{greek}µ\selectlanguage{english}m) & 7.46 (\selectlanguage{ngerman}\textbf{±}1.64) &
7.63 (\selectlanguage{ngerman}\textbf{±}1.18) & -\tabularnewline
Hydraulic diameter (D\textsubscript{h} - \selectlanguage{greek}µ\selectlanguage{english}m) & \textbf{38.20}
(\selectlanguage{ngerman}\textbf{±}6.17) & \selectlanguage{ngerman}\textbf{77.70 (±8.23)} & \textbf{103.40
*}\tabularnewline
Vessel density (V\textsubscript{d} -- v mm\textsuperscript{-2}) & 81.90
(\selectlanguage{ngerman}\textbf{±}30.39) & 122 (\selectlanguage{ngerman}\textbf{±}35.74) & -\tabularnewline
Vessel grouping index (V\textsubscript{g}) & \textbf{2.42}
(\selectlanguage{ngerman}\textbf{±}0.58) & \textbf{3.63} (\selectlanguage{ngerman}\textbf{±}0.53) & \textbf{50.00
**}\tabularnewline
Vessel element high (\selectlanguage{greek}µ\selectlanguage{english}m) & 335.3 (\selectlanguage{ngerman}\textbf{±}102.27) & 327.5
(\selectlanguage{ngerman}\textbf{±}72.50) & -\tabularnewline
Pit diameter (\selectlanguage{greek}µ\selectlanguage{english}m) & \textbf{4.28} (\selectlanguage{ngerman}\textbf{±}0.72) & \textbf{4.95}
(\selectlanguage{ngerman}\textbf{±}0.79) & \textbf{15.65 ***}\tabularnewline
Arrangement & Radial & - &\tabularnewline
Specific conductivity (K\textsubscript{p} -- kg m\textsuperscript{-1}
Mpa\textsuperscript{-1} s\textsuperscript{-1} 10\textsuperscript{-6}) &
\textbf{4.52} (\selectlanguage{ngerman}\textbf{±}2.70) & \textbf{106.98} (\selectlanguage{ngerman}\textbf{±}31.23) &
\textbf{2262,81 **}\tabularnewline
\textbf{Fibers} & & &\tabularnewline
Length (\selectlanguage{greek}µ\selectlanguage{english}m) & \textbf{721.8} (\selectlanguage{ngerman}\textbf{±}175.90) & \textbf{661.9}
(\selectlanguage{ngerman}\textbf{±}124.6) & \textbf{-8.30 ***}\tabularnewline
Wall thickness (\selectlanguage{greek}µ\selectlanguage{english}m) & \textbf{1.87} (\selectlanguage{ngerman}\textbf{±}0.38) & \textbf{2.41}
(\selectlanguage{ngerman}\textbf{±}0.56) & \textbf{28.88 ***}\tabularnewline
Lumen area (\selectlanguage{greek}µ\selectlanguage{english}m) & \textbf{58.6} (\selectlanguage{ngerman}\textbf{±}33.17) & \textbf{74.3}
(\selectlanguage{ngerman}\textbf{±}38.65) & \textbf{26.79 ***}\tabularnewline
\textbf{Rays} & & &\tabularnewline
Width & Unisseriate & Uni and biseriate &\tabularnewline
Cellular composition & Upright and square & Predominantly procumbent and
square &\tabularnewline
\bottomrule
\end{longtable}

Measurements were taken at 4/5 of the stem length towards the base of
the stem; means and standard deviations are presented. Differences (\%)
were calculated using the formula ((L - Ss) Ss\textsuperscript{-1})\selectlanguage{ngerman}×100.
Significant differences are marked in bold. *: p \textless{}0.05; **: p
\textless{}0.01; ***: p \textless{}0.001.

\textbf{The Presence of Physical Supports Triggers Lianescent Xylem
Formation}

To investigate whether the attachment to physical supports triggers the
formation of lianescent xylem, we analyzed the secondary xylem anatomy
of the longest stems of five supported and five unsupported plants at
regular intervals (Fig. 4). Supported plants had longer (6.6 ± 1.6
{[}SD{]}) m) and thicker stems (10.4 ± 3.6 mm) compared to unsupported
plants (2.7 ±0.8 m long and 9.4 ± 0.8 mm thick).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig. 4.} Stem cross sections of supported (a, c, and e) and
unsupported plants (b, d, and f) at different distances from the shoot
apex: one meter (a and b), two meters (c and d), and three meters (e and
f). Lianescent xylem is produced only in supported plants (white
arrows), from 1.5 meters from the apex along the proximal portion of the
stem (c and e). In contrast, only self-supporting xylem is produced by
unsupported plants (d and f). Note the smaller xylem production in
supported plants, measured by the distance between the pith (P, not
shown in d and f) and the cambium (C). Scale: 30 \selectlanguage{greek}µ\selectlanguage{english}m. Unstained sections.

All plants produced self-supporting xylem near the stem apex (Fig. 5a
and b), while the production of lianescent xylem was observed only in
plants grown with physical supports. Lianescent xylem formation started
from 1.5 -- 2 m from the apex and extended through the proximal portion
of the stem in supported plants (Fig. 5c and 5e). In contrast,
unsupported plants only produced self-supporting xylem along the entire
stem (Fig. 5d and 5f).

The production of lianescent xylem was associated with a smaller xylem
production, measured as the linear distance between the cambium and the
beginning of the secondary xylem (Fig. 5). In contrast, unsupported
plants, which only formed the self-supporting xylem, produced more
secondary xylem at the same distance from the apex compared to supported
plants

These findings indicate that lianescent xylem formation occurs in
response to stem attachment to physical supports, leading to reduced
cambial activity, increased vessel size, and enhanced xylem
conductivity.

\emph{B. magnifica} \textbf{Cambium and Differentiating Xylem
Transcriptome is Consistent with Model Tree Species}

To understand the transcriptional regulation underlying the concomitant
formation of distinct xylem phases in different parts of the same plant,
we conducted an RNA-Seq analysis of the active cambium and
differentiating xylem of portions producing the self-supporting or the
lianescent xylem. We sequenced six biological samples for each phase,
resulting in approximately 512 million paired-end reads (40 GB,
Supporting Information Fig. S3, Table S1a). \emph{De novo} assembly
produced 133,883 transcripts, from which 54,207 longest isoforms were
recovered (Supporting Information Fig. S3). Among these, 20,548
transcripts showed BLASTX hits against Viridiplantae sequences in the
SwissProt database (downloaded on September 18, 2019). We identified
20,428 sequences associated with Gene Ontology (GO) terms, which were
further analyzed (Supporting Information Fig. S3 and Tables S1a, S1b,
S1c). Out of the 7,198 unique GO terms identified, 3,738, 2,714, and 746
were related to biological processes (BP), molecular functions (MF), and
cellular components (CC), respectively (Supporting Information Tables
S2).

The completeness of the assembled transcriptome was assessed using BUSCO
v5.2.2 (Manni \emph{et al.} , 2021), which showed 99\% complete BUSCOs
(number of BUSCOs: 425), indicating the high quality of our assembly.
Among the 20,428 GO-associated sequences, we identified two transcripts
homologous to the cambial markers PHLOEM INTERCALATED WITH XYLEM
(PXY)/TDIF RECEPTOR (TDR; DN4695\_c0\_g1\_i3 and DN19385\_c0\_g1\_i1;
transcript identifiers listed in Supporting Information Table S1b) and
one homologous to WUSCHEL RELATED HOMEOBOX4 (WOX4; DN4743\_c0\_g1\_i1)
(Hirakawa \emph{et al.} , 2010; Suer \emph{et al.} , 2011; Shi \emph{et
al.} , 2019; Shi \emph{et al.} , 2021). We also identified the phloem
markers ALTERED PHLOEM DEVELOPMENT (APL; DN4491\_c0\_g1\_i2 and
DN5859\_c0\_g1\_i6) and NAC domain-containing protein 86 (NAC086;
DN12555\_c0\_g1\_i1) (Froelich \emph{et al.} , 2011; Furuta \emph{et
al.} , 2014; Shi \emph{et al.} , 2021), indicating successful sampling
of cambium and differentiating xylem tissues.

To check the quality of the assembled transcriptome, we compared the
functional annotation of \emph{B. magnifica} with those of the secondary
xylem-forming tissues from model species \emph{Eucalyptus grandis}
and\selectlanguage{ngerman}\emph{Populus × euramericana,} following the approach of
Zinkgraf\emph{et al.} (2017) using the data generated by Xu \emph{et al}
. (2014). The cambium and differentiating xylem transcriptome of B.
magnifica (20,428 transcripts) exhibited a higher number of transcripts
with associated GO terms compared to the other two species (15,127 and
14,186 for \emph{E. grandis} and \emph{P. × euramericana} ,
respectively). Interestingly, all the most significant BP present in the
model species were also found in \emph{B. magnifica} , with a similar
number of associated transcripts, whether considering the total
transcriptome (\emph{i.e.} , from both phases combined, Fig. 5a) or each
phase separately. Additionally, we identified 105 BP exclusively present
in\emph{B. magnifica} (Supporting Information Fig. S4), potentially
encompassing transcripts with unique functions in \emph{B. magnifica.}

\textbf{The Transcriptome of Self-Supporting and Lianescent Phases of
Xylem Development Differs in Their Most Relevant Gene Functions}

To identify differentially expressed genes (DEGs) between the
self-supporting and lianescent phases, we assessed the diversity of
expression profiles within each phase. A multidimensional scaling (MDS)
analysis showed a clear grouping of samples within each set, indicating
their suitability for further analysis (Supporting Information Fig. S5).

Applying a significance cut-off of p \textless{} 0.05, false discovery
rate (FDR) \textless{} 0.05, and logFC \textgreater{}
\textbar{}2\textbar{} (Fig. 5b), we identified 140 upregulated
transcripts in the self-supporting phase and 85 upregulated transcripts
in the lianescent phases (Supporting Information Tables S3a, c,
respectively; count of annotated transcripts shown in Supporting
Information Table S1c). Enrichment analysis revealed that in the
self-supporting phase, the most represented GO categories (BP, MF, and
CC) could be grouped into cell wall-related processes (overrepresented),
transcriptional regulation, and protein metabolism (both
underrepresented) (Supporting Information Table S3b). In the lianescent
phase, the BP category \emph{response to stimulus} , which
includes\emph{response to} \emph{organic substance} , \emph{response to
hormone,}and \emph{response to endogenous stimulus} , was highly
overrepresented, suggesting the involvement of hormone signaling in
xylem differentiation. The MF \emph{L-glutamine transmembrane
transporter activity} was also overrepresented (Supporting Information
Table S3d).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Fig. 5.} (a) Comparative functional annotation of cambial
transcriptomes from\emph{B. magnifica} (Bma) and the model species
\emph{Eucalyptus grandis} (Egr) and \selectlanguage{ngerman}\emph{Populus × euramericana} (Pop)
(using data generated by Xu \emph{et al.} 2014). (b) Volcano plot
showing transcript expression p-value versus log2 of fold-change of
240,428 annotated transcripts associated with GO terms. A total of 225
differentially expressed genes (DEGs) were recovered using a cutoff of p
\textless{} 0.05 and log2 fold change \textgreater{}
\textbar{}2\textbar{}: 140 up-regulated in self-supporting xylem forming
tissue (blue dots) and 85 up-regulated in lianescent xylem forming
tissue (green dots). (c) The five most represented categories of
manually curated annotation of the 225 DEGs. In total, 154 (68\%) DEGs
were annotated in these five categories. Histograms in (d), (e), and (f)
show the expression fold changes of genes related to cell wall, cell
division, and hormone response, respectively. \textbf{MYB26/52} : MYB
DOMAIN PROTEIN 26/52;\textbf{NC104} : NAC DOMAIN-CONTAINING PROTEIN 104/
XYLEM NAC DOMAIN 1;\textbf{MYBH} : MYB HYPOCOTYL ELONGATION-RELATED;
\textbf{CESA7} : CELLULOSE SYNTHASE CATALYTIC SUBUNIT 7;
\textbf{FLA1/9/11/12} : FASCICLIN-LIKE ARABINOGALACTAN 1/9/11/12;
\textbf{SP1L2/5} : SPIRAL1-like 2/5; \textbf{CAD6/7} : CINNAMYL ALCOHOL
DEHYDROGENASE 6/7;\textbf{CYP75B1} : FLAVONOID 3'-MONOOXYGENASE;
\textbf{CHS} : CHALCONE SYNTHASE; \textbf{CYCB2;3} : CYCLIN B2;3;
\textbf{TCP20} : TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL
FACTOR1-20; \textbf{MAIL1} : MAINTENANCE OF MERISTEMS-LIKE1;
\textbf{KNAT6} : HOMEOBOX PROTEIN KNOTTED-1-LIKE6; \textbf{PTL} : PETAL
LOSS; \textbf{CKX1/5} : CYTOKININ OXIDASE/DEHYDROGENASE 1/5;
\textbf{UGT85A1/85ª3} : UDP-GLUCOSYL TRANSFERASE 85ª1/85ª3;
\textbf{GAOX1} : GA20 OXIDASE 1; \textbf{CYP90B1} : CYTOCHROME P450
90B1; \textbf{KIN14Q/7E/5B/12D} : KINESIN-LIKE-14Q/7E/5B/12D;
\textbf{JASON} ; \textbf{ASY1} : ASYNAPTIC 1;\textbf{SGO2} : SHUGOSHIN
2; \textbf{HMGB13} : 3XHIGH MOBILITY GROUP-BOX1;\textbf{OS1} : PARALLEL
SPINDLE 1; \textbf{PIN1} : PIN-FORMED 1;\textbf{IAA19} ; \textbf{ERF1B}
: ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR 1B; \textbf{LSH10} :
LIGHT-DEPENDENT SHORT HYPOCOTYLS 10; \textbf{HB17} : HOMEOBOX-LEUCINE
ZIPPER PROTEIN 17; \textbf{BLH1} : BEL1-LIKE HOMEODOMAIN; \textbf{EDL3}
: EID1-LIKE F-BOX PROTEIN 3; \textbf{NPF4} : NRT1/ PTR FAMILY 4;
\textbf{HAB2} : PROTEIN PHOSPHATASE 2C;\textbf{GAT22} : GATA
TRANSCRIPTION FACTOR 22.

\textbf{Upregulation of Cell Division and Cell Wall-Related Transcripts
Characterizes the Self-Supporting Phase Transcriptome}

To better understand the gene functions that determined the differences
between self-supporting and lianescent xylem differentiation, we
performed a manually-curated comprehensive annotation of all DEGs
against the literature (Supporting Information Table S3a, c, column J).
In line with the higher number of cells per area and the consequent
greater cell wall deposition in self-supporting xylem, 11 \% and 30 \%
of the DEGs are associated with cell division and cell wall, contrasting
with only 4 \% and 18 \% in the lianescent counterpart, respectively
(Fig. 5c, Supporting Information Table S4).

Among the DEGs associated with cell division in the self-supporting
phase, we found two transcripts homologous to positive regulators of the
cell cycle, the mitotic-specific cyclin CYCB2;3 and the transcription
factor (TF) TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20
(\emph{TCP20} ) (DN860\_c0\_g2\_i2 and DN10604\_c0\_g1\_i2,
respectively; Fig. 5e). We also found two transcripts whose homologs are
related to meristem identity and activity maintenance, a homolog of the
nuclear protein MAINTENANCE OF MERISTEMS-LIKE1 (MAIL1,
DN8697\_c0\_g1\_i3), and a homolog to the TF HOMEOBOX PROTEIN
KNOTTED-1-LIKE6 (KNAT6, DN15272\_c0\_g1\_i2), besides four mitotic
specific kinesin homologs (KIN14Q, KIN7E, KIN5B, KIN12D;
DN13653\_c1\_g1\_i1, DN12012\_c0\_g1\_i1, DN11506\_c0\_g1\_i1, and
DN13654\_c0\_g1\_i2, respectively) and five chromatin segregation
related proteins (JASON, ASY1, SGO2, HMGB13, PS1; DN1459\_c0\_g2\_i1,
DN5155\_c0\_g1\_i1, DN19359\_c0\_g1\_i1, DN5075\_c0\_g1\_i1, and
DN2177\_c0\_g1\_i1, respectively; Fig. 5e).

Two transcripts homologs to \emph{MYB} TFs involved in the regulation of
SCW biosynthesis, \emph{MYB26,} and \emph{MYB52} , were upregulated in
the self-supporting phase (DN7293\_c0\_g2\_i4 and DN35518\_c0\_g1\_i1
respectively; Fig. 5d). Forty upregulated transcripts are directly
involved in the SCW biosynthesis and structural modification, among
which we highlight homologs of cellulose synthase A (\emph{CESA7} ,
DN8977\_c0\_g2\_i4), four fasciclin-like arabinogalactan homolog
proteins (FLA1, FLA9, FLA11, FLA12; DN7969\_c0\_g1\_i2,
DN10033\_c0\_g1\_i1, DN49576\_c0\_g1\_i1, and DN52901\_c0\_g1\_i1,
respectively), and two SPIRAL1-like (SP1L2 and SP1L5, DN1885\_c0\_g1\_i2
and DN7226\_c0\_g1\_i1 respectively; Fig. 5d).

\textbf{Upregulation of Response to Stimulus, Hormone-Responsive, and
Defense/Cell Death-Related Transcripts Characterizes Lianescent Phase
Transcriptome}

Manually-curated annotation of the lianescent phase transcriptome showed
a higher proportion of TF and hormone-responsive genes, respectively, 17
\% and 30 \% of all DEGs of this phase, in contrast to only 11 \% and 4
\% in the self-supporting counterpart. Additionally, 25 \% of the DEGs
identified in the lianescent phase correspond to genes involved in
defense/cell death mechanisms, compared to only 18 \% of the DEGs in
this category in the self-supporting phase (Fig. 5c, Supporting
Information Table S4).

Our analysis revealed important cell differentiation regulators homologs
among the upregulated DEGs that may be responsible for the fewer cell
divisions and the production of larger vessels in the lianescent xylem.
The repressor of cambial cell proliferation PETAL LOSS (PTL; Fig. 5e)
and NAC DOMAIN-CONTAINING PROTEIN 104/ XYLEM NAC DOMAIN 1 (NC104; Fig.
5d), which delay SCW production and programmed cell death (PCD), had
homologs upregulated in this phase (DN52789\_c0\_g1\_i1 and
DN10071\_c0\_g1\_i1, respectively). \emph{ACAULIS5} (\emph{ACL5} ) was
another gene responsible for preventing premature PCD with an
upregulated homolog in the lianescent phase, although with a logFC of
1.1. Another candidate TF that may be related to large vessel formation
was DN31217\_c0\_g1\_i1, a homolog of the \emph{A. thaliana} MYB
HYPOCOTYL ELONGATION-RELATED (MYBH; Fig. 5d), that induces cellular
growth triggered by auxin accumulation.

Fourteen upregulated transcripts have been annotated as TF homologs, six
described as hormone-responsive: IAA19 (DN53387\_c0\_g1\_i1), GATA
TRANSCRIPTION FACTOR 22 (GAT22, DN16098\_c0\_g1\_i1),
ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR 1B (ERF1B,
DN35952\_c0\_g1\_i1), LIGHT-DEPENDENT SHORT HYPOCOTYLS 10 (LSH10,
DN507\_c0\_g1\_i1), HOMEOBOX-LEUCINE ZIPPER PROTEIN 17 (HB17,
DN11122\_c0\_g1\_i1), and BEL1-LIKE HOMEODOMAIN (BLH1,
DN13026\_c0\_g1\_i2). These TFs are responsive to indole-3-acetic acid
(IAA), cytokinin (CK), ethylene (ET), brassinosteroid (BR), and abscisic
acid (ABA, HB17 and BLH1), respectively (Fig. 5f).

Not only hormone-responsive TF homologs were upregulated, but also
homologs to transcripts related to hormone metabolism and signaling
pathways. CK was the hormone with the highest number of annotated
transcripts related to its catabolism/deactivation: two cytokinin
dehydrogenases and two UDP-glycosyltransferases homologs (CKX1, CKX5,
UGT85A1, UGT85A3; DN20233\_c0\_g1\_i1, DN9178\_c0\_g1\_i1,
DN17065\_c0\_g1\_i1, DN49211\_c0\_g1\_i1 respectively). Upregulation of
homologs to BR and gibberellin (GA) biosynthetic genes were also
identified, CYTOCHROME P450 90B1 and GA20 OXIDASE 1 (GAOX1) respectively
(DN4579\_c0\_g1\_i1, DN11261\_c0\_g1\_i1; Fig. 5e), as well as three
genes that participate in the ABA signaling pathway, EID1-LIKE F-BOX
PROTEIN 3 (\emph{EDL3} ), ABA-IMPORTING TRANSPORTER 1 (\emph{AIT1} ) and
HOMOLOGY TO ABI2 (\emph{HAB2} ) (DN45297\_c0\_g1\_i1,
DN10749\_c0\_g1\_i1, and DN10194\_c0\_g1\_i5, respectively). Another
highlight was the upregulation of the auxin efflux carrier PIN1 homolog
(DN10775\_c0\_g1\_i1; Fig. 5f), showing the relevance of all major plant
hormones in the lianescent xylem differentiation process.

Together, these results show the prevalence of cell division and SCW
biosynthesis during the formation of self-supporting xylem and the more
complex regulation of the lianescent xylem formation, with the
predominance of TF and hormone-responsive genes in this phase.

\textbf{Discussion}

In the present work, we characterize how the attachment to physical
support leads to the production of the lianescent xylem in the liana
species \emph{Bignonia magnifica} . We have also demonstrated that the
more complex anatomy of the lianescent xylem results from a more complex
transcriptional regulation involving the upregulation of transcriptional
factors and hormone-responsive genes.

Our finding that the attachment to physical supports signals the onset
of lianescent xylem production in \emph{B. magnifica} (Bignoniaceae), a
species that employs leaflets modified into tendrils to climb, is
similar to that recently observed in the also tendrilate
liana\emph{Serjania Mexicana} (Sapindaceae; Rajput \emph{et al.} 2021)
and in the stem twinner \emph{Condylocarpon guianense} (Apocynaceae;
Soffiatti et al., 2022). This mechanical and functional convergence in
lianas from different families and with different climbing strategies
highlights the relevance of the environmental perception in the habit.
The attachment to external supports relaxes the initial mechanical
function of the xylem, representing an important resource economy in the
construction of supportive tissue for lianas and allowing the
specialization in hydraulic conduction. The phenotypic plasticity
represented by the retention of the mechanical function in young
searcher stems and the control of lianescent xylem formation only by
stems attached to external supports, in turn, allows to better explore
the environment and can be viewed as an important ecological and
evolutionary strategy, considering the high prevalence of this character
among lianas (Caballé, 1993, 1998).

The \emph{ca.} 30\% reduction of fibers relative area represented the
replacement of this cell type by large vessels, stressing the hydraulic
conduction specialization (Ewers\emph{et al.} , 1989, 1990; Ewers and
Fisher 1991; Gasson and Dobbins, 1991; Fisher and Ewers, 1995, Lens
\emph{et al.} ,2011) over the mechanical function. Pit diameter increase
might enhance hydraulic conductivity as well, as pit conductivity
resistance accounts for about 80\% of the total resistance (Choat et
al., 2006), while it might also increase the probability of embolism
spread (Wheeler et al. 2005; Choat et al. 2005; Hacke et al. 2006).
Similarly, the connectivity of the vessel network, as indicated by the
vessel grouping index, increases pathway redundancy and conductivity
safety (Carlquist, 1985; Ewers\emph{et al.} , 2007; Wason \emph{et al.}
, 2021). However, this trend is limited, as enhanced redundancy can be
counteracted by the facilitated spread of embolism through the
interconnected vessels above a certain threshold (Mrad et al. 2021).
Finally, the increase in fiber wall thickness was in contrast to what
was found in other lianas (Gartner, 1991; Ménard \emph{et al.} , 2009),
but it may have a role in preventing embolisms, since volume allocation
to fiber walls increases embolism resistance (Janssen \emph{et al.} ,
2020). Our findings highlight the complex relationship between
anatomical characteristics, hydraulic conductivity, and gene expression,
providing insights into the differentiation of self-supporting and
lianescent xylems.

The self-supporting and lianescent xylems showed distinct expression
patterns. We have visually summarized the main findings of our
differential expression analysis, establishing correlations between them
and the observed anatomical changes during the transition from the
self-supporting to lianescent phase in \emph{B. magnifica} xylem (Fig.
6). The self-supporting xylem shows a higher level of cellular division,
evidenced by the increased secondary xylem production and a composition
comprising fibers and small diameter vessels, resulting in a greater
cell density per unit area. This pattern is in accordance with the
upregulation of homologs of the mitotic-specific cyclin
\emph{CYCB2;3}(Van Leene \emph{et al.} 2010) and of the cell cycle
regulator\emph{TCP20} (Guan \emph{et al.} 2017), increasing cambial cell
divisions in this phase. In this sense, the upregulation of homologs of
mitotic-specific kinesins and chromatin segregation-related proteins
were also identified. On the other hand, the lianescent-phase
transcriptome showed the upregulation of a homolog of the cambial cell
proliferation repressor \emph{PTL} (Zhang \emph{et al.} , 2019), and
four different CK catabolism/deactivation homolog transcripts, CKX1/5
and UGT85A1/85A3. CKs have long been known to induce cambial activity
(Torrey \& Loomis, 1967; Aloni \emph{et al.} , 1990), and reduced
concentrations of bioactive CKs result in decreased secondary growth in
both \emph{Arabidopsis} and \emph{Populus} (Matsumoto-Kitano \emph{et
al.} , 2008; Nieminen \emph{et al.} , 2008).

The higher number of cells in the self-supporting phase is expected to
correlate with more abundant cell wall biosynthesis. In this sense,
homologs of master MYB TFs, \emph{MYB26} and \emph{MYB52} , involved in
the regulation of SCW biosynthesis (Yang \emph{et al.} , 2007, 2017;
Cassan-Wang \emph{et al.} , 2013), were found to be upregulated in the
self-supporting phase. This upregulation was accompanied by the higher
expressions of cellulose synthase A homologs and four fasciclin-like
arabinogalactan proteins that have been proposed to contribute to stem
strength and stiffness in \emph{Eucalyptus} and \emph{A. thaliana} by
increasing cellulose deposition and affecting cell-wall matrix integrity
(Macmillan \emph{et al.} , 2010). This expression pattern is compatible
with the higher stiffness of young liana stems and with the colonizing
role of searcher branches in nature (Caballé, 1993, 1998; Rowe \& Speck,
1996, 2005; Soffiatti \emph{et al.} , 2022). Interestingly,
two\emph{SPR1} homolog transcripts required for the anisotropic cell
growth (Nakajima \emph{et al} ., 2004) were upregulated in the
self-supporting phase. \emph{SPR1} overexpression increases cell
elongation, in accordance with the longer fibers found in the
self-supporting xylem, while \emph{spr} loss-of-function mutants show
helical growth of epidermal cells and entire organs (Smyth, 2016), a
characteristic of twining vine stems that has long been known (Darwin,
1875; Isnard \& Silk, 2009).

The most striking feature of the lianescent xylem is the production of
large vessel elements, which dramatically increased potential specific
conductivity and vessel relative area. The transcriptional regulation of
cell death is an essential aspect of the overall xylem maturation
program, which also encompasses cell expansion and SCW deposition, and
emphasizes the crucial role of inhibiting cell death during this process
(Bollhöner\emph{et al} ., 2012). Besides the upregulation of NC104/XND1,
which represses cell wall production and PCD (Zhang \emph{et al} .,
2020; Zhong\emph{et al.} , 2021), ACAULIS5 (ACL5), another gene
responsible for preventing premature PCD (Muñiz \emph{et al} ., 2008),
was upregulated in the lianescent phase. The increase in cell expansion
period before PCD was linearly correlated with the lumen area in
\emph{Picea} trees tracheids (Anfodillo \emph{et al} ., 2013; Buttò
\emph{et al.} , 2019). Yet, a homolog of MYBH TF, which was shown to
trigger cell growth through IAA accumulation (Kwon \emph{et al} ., 2013,
Lu \emph{et al} ., 2014), was also upregulated in the lianescent phase
and might also be involved in the differentiation of larger vessels.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\par\null

\textbf{Fig. 6.}Transcriptional regulatory network model for the
self-supporting and lianescent xylem formation in the stems of the liana
\emph{B. magnifica}based on differential gene expression results and
comprehensive anatomical analysis. The self-supporting phase has a
higher growth in thickness and is composed mainly of fibers and small
vessels arranged in radial files in front of protoxylem poles.
Self-supporting xylem formation is associated with a higher expression
of cell division and secondary cell wall biosynthesis-related
transcripts. The lianescent phase has slower growth in thickness and is
composed of a smaller fraction of fibers and a higher proportion of
vessels. This change is due to the production of large vessels, which
are produced throughout the cambium circumference. The formation of
lianescent xylem is linked to a much more complex transcription network
involving transcription factors, hormone-responsive genes, delayed
programmed cell death, and redistribution of auxin-mediated by PIN
proteins. Capital letters: observed morphoanatomical characters; in red:
deduced characters.\textbf{CYCB2;3} : CYCLIN B2;3; \textbf{TCP20} :
TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20;
\textbf{MAIL1} : MAINTENANCE OF MERISTEMS-LIKE1; \textbf{KNAT6} :
HOMEOBOX PROTEIN KNOTTED-1-LIKE6; \textbf{KIN14Q/7E/5B/12D} :
KINESIN-LIKE-14Q/7E/5B/12D;\textbf{JASON} ; \textbf{ASY1} : ASYNAPTIC 1;
\textbf{SGO2} : SHUGOSHIN 2;\textbf{HMGB13} : 3XHIGH MOBILITY
GROUP-BOX1; \textbf{PS1} : PARALLEL SPINDLE 1; \textbf{MYB26/52} : MYB
DOMAIN PROTEIN 26/52; \textbf{CESA7} : CELLULOSE SYNTHASE CATALYTIC
SUBUNIT 7; \textbf{FLA1/9/11/12} : FASCICLIN-LIKE ARABINOGALACTAN
1/9/11/12; \textbf{SP1L2/5} : SPIRAL1-LIKE2/5; \textbf{PTL} : PETAL
LOSS; \textbf{CKX1/5} : CYTOKININ OXIDASE/DEHYDROGENASE 1/5;
\textbf{UGT85A1/85A3} : UDP-GLUCOSYL TRANSFERASE 85A1/85A3;
\textbf{GAOX1} : GA20 OXIDASE 1; \textbf{C90B1} : CYTOCHROME P450 90B1;
\textbf{PIN1} : PIN-FORMED 1; \textbf{ACL5} : ACAULIS5;
\textbf{NC104/XND1} : NAC DOMAIN-CONTAINING PROTEIN 104/ XYLEM NAC
DOMAIN 1; \textbf{MYBH} : MYB HYPOCOTYL ELONGATION-RELATED;\textbf{HB17}
: HOMEOBOX-LEUCINE ZIPPER PROTEIN 17; \textbf{BLH1} : BEL1-LIKE
HOMEODOMAIN; \textbf{IAA19} ; \textbf{GAT22} : GATA TRANSCRIPTION FACTOR
22; \textbf{ERF1B} : ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR 1B;
\textbf{LSH10} : LIGHT-DEPENDENT SHORT HYPOCOTYLS 10.

IAA polar flow also seems to be modified in the lianescent phase, as
indicated by the upregulation of the polar auxin transporter PINFORMED1
(PIN1). Various processes take place along a PIN1-driven auxin flow,
including bundle differentiation (Sachs, 1981; Scarpella \emph{et al} .,
2006), cambium formation and maintenance (Snow,1935; Uggla \emph{et al}
., 1996; Ko \emph{et al} ., 2004; Mazur \emph{et al} ., 2014; Ye \&
Zhong, 2015), and the differentiation of vessels bypassing wounds
(Mazur\emph{et al} ., 2016). Hence, vascular differentiation can be used
as a marker of auxin flow (Sachs, 2000). Similarly, vessels in the
secondary xylem are formed by the coordinated differentiation of
thousands of cells, \emph{i.e.} the vessel elements, in a continuous
longitudinal series that can reach several meters in lianas (Zimmermann
\& Jeje, 1981; Ewers \emph{et al.} , 1990). PIN1 upregulation is likely
related to the higher number of xylem mother cell derivatives
differentiating into vessel elements, leading to the broader
distribution of vessels observed in the lianescent xylem. This result
reinforces those previously reported suggesting that polar auxin
transport defines vessel distribution and size in the secondary xylem
(Johnson \emph{et al} ., 2018; Novitskaya \emph{et al.} , 2020).

Transcription factors are central nodes in the transcriptional network
responsible for cell identity and differentiation in the xylem
(Xie\emph{et al.} , 2021). Here we found 14 TF upregulated in the
lianescent phase, six of which participate in hormone-response pathways:
two homologs to ABA response regulators, \emph{HB17} and \emph{BLH1} ,
which also responds to CK (Hoth \emph{et al.} , 2003; Park \emph{et al.}
, 2013); a CYTOKININ-RESPONSIVE GATA FACTOR 1 (\emph{GAT22} ) homolog,
which was shown to have a regulatory role downstream from both IAA and
GA signaling pathways (Richter \emph{et al.} , 2013); a homolog to
ethylene-responsive transcription factor ERF1B, which was found to
increase IAA production (Mao \emph{et al} . 2016) and integrates ET and
JA pathways (Lorenzo \emph{et al.} 2003); a homolog to the IAA repressor
IAA19; and a homolog to the BR responsive TF \emph{LSH10} , which was
recently found to be a co-repressor of target genes by its epigenetic
regulation (Goda \emph{et al.} , 2004; Vo Phan \emph{et al.} , 2023).

The crosstalk between TFs and various plant hormones in lianescent xylem
differentiation was further supported by the upregulation of gene
homologs associated with hormone biosynthesis and signaling pathway.
Homologs to GAOX1 and C90B1, responsible for GA and BR biosynthesis,
respectively, were upregulated in this phase. GA is known to act
synergistically with IAA, increasing\emph{PIN1} expression and polar
auxin transport (Willige \emph{et al.} , 2011; M\selectlanguage{ngerman}äkilä \emph{et al.} ,
2023), conserving cambium homeostasis and xylem differentiation
(Ben-Targem \emph{et al.} , 2021), and induces the expression of the
same genes as auxin feeding experiments in\emph{Populus} (Bjorklund
\emph{et al} ., 2007). Similarly, BR regulates PIN protein expression
and high levels of IAA signaling in the cambium (Li \emph{et al.} ,
2005; Lee \emph{et al.} , 2021), cellular differentiation and SCW
biosynthesis (Yamamoto \emph{et al.} , 1997; Caño-Delgado \emph{et al.}
, 2004; Du \emph{et al.} , 2020), and affects CK biosynthesis and
catabolism genes (Wang \emph{et al.} , 2022). ABA, which is known as a
crucial hormone to drought stress response, was recently shown to
regulate the SCW formation under drought conditions (Yu \emph{et al.} ,
2021; Liu \emph{et al.} , 2021), besides modulating the expression of
target genes that affect vessel traits by inducing the expression of the
AREB1 TF (Li \emph{et al} , 2019). Two ABA signaling pathway regulators,
HAB2 and EDL3 (Saez \emph{et al.} , 2004; Yoshida\emph{et al.} , 2006;
Koops \emph{et al.} , 2011) were upregulated in the lianescent phase,
what may indicate the cooption of the drought response pathway for the
higher range of vessel diameters produced in the lianescent phase.

\textbf{Concluding Remarks}

In the present study, we verified the profound impacts of physical
support on the liana \emph{B. magnifica} wood anatomy, promoting the
formation of the lianescent xylem. The detailed characterization of
xylem anatomy showed that the onset of the lianescent phase is
characterized by the beginning of vessel production also by the
interfascicular cambium, previously restricted to the fascicular
cambium; by the formation of large vessels of a new diameter class, that
drastically increases potential specific conductivity; and by a lesser
amount of cambial divisions. The comprehensive integration of anatomical
and differential expression analysis data allowed us to propose a model
to characterize the molecular control of the lianescent vascular
syndrome establishment (Fig. 6). Our model shows that the more complex
lianescent xylem reflects a more intricate transcriptional regulation
network, involving a more diverse repertoire of TFs and
hormone-responsive genes. We hope the analysis of the transcriptional
control of the vascular system differentiation in lianas helps to better
understand the differentiation of the vascular system in other habits,
in addition to shedding light on the formation of the structural
diversity present in tropical forests, widely recognized as a center of
global diversity.

\textbf{Acknowledgments}

We thank Dr. Paula Elbl for critical inputs to this study and Dr.
Horácio Montenegro for his contribution to computational infrastructure.
This study was supported by the São Paulo Research Foundation --- FAPESP
(Process 18/06917-7), by the Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior -- Brasil (CAPES) -- Finance Code 001, and by The
National Council for Scientific and Technological Development --- CNPq
(process 142223/2015-8).

\textbf{Author contribution}

ACL and VA conceived the project. ACL and MR designed the experiments.
ACL, CSG, and VA analyzed and interpreted anatomical data. Sequence
analyses were carried out by ACL, DTS, and SCSA. LLC granted access to
the computational facility and was responsible for data curation. ACL
and MR interpreted differential gene expression analysis (DEG) results
and conducted the hand-curated annotation of DEGs. ACL wrote the article
with inputs from MR, CSG, SCSA, and VA. All authors read and approved
the final manuscript.

\textbf{Data availability}

The raw reads from the 12 samples used in this study are publicly
available at the Sequence Read Archive (NCBI-SRA), bioproject accession
number PRJNA927333 (submitted on 01/25/2023).

\textbf{References}

\textbf{Allona I, Quinn M, Shoop E, Swope K, Cyr SS, Carlis J, Riedl J,
Retzel E, Campbell Mm, Sederoff R, Whetten RW. 1998.} Analysis of xylem
formation in pine by cDNA sequencing. \emph{Proceedings of the National
Academy of Sciences}\textbf{95:} 9693-9698.

\textbf{Aloni R, Baum SF, Peterson CA. 1990.} The role of cytokinin in
sieve tube regeneration and callose production in wounded Coleus
internodes. \emph{Plant Physiology} \textbf{93:} 982--989.

\textbf{Anfodillo T, Petit G, Crivellaro A. 2013.} Axial conduit
widening in woody species: a still neglected anatomical
pattern.~\emph{Iawa Journal} ~\textbf{34:} 352-364.

\textbf{Angyalossy V, Pace MR, Lima AC. 2015.} Liana anatomy: a broad
perspective on structural evolution of the vascular system. In Schnitzer
SA, Bongers F, Burnham RJ, Putz FE, eds. \emph{Ecology of Lianas} .
Chichester, UK: John Wiley \& Sons Ltd, 253-287.

\textbf{Baas P, Ewers FW, Davis SD, Wheeler EA. 2004.} Evolution of
xylem physiology. In Hemsley AR, Poole I, eds. \emph{The Evolution of
Plant Physiology} . London, UK: Elsevier Academic Press: 273-395.

\textbf{Barbosa ACF, Pace MR, Witovsk L, Angyalossy V. 2010.} A new
method to obtain good anatomical slides of heterogeneous plant
parts.\emph{IAWA Journal} \textbf{31:} 373-383.

\textbf{Beeckman H. 2016.} Wood anatomy and trait-based
ecology.\emph{IAWA journal} ~\textbf{37:} 127-151.

\textbf{Ben-Targem M, Ripper D, Bayer M, Ragni L. 2021.} Auxin and
gibberellin signaling cross-talk promotes hypocotyl xylem expansion and
cambium homeostasis. \emph{Journal of Experimental Botany}
\textbf{72:}3647-3660.

\textbf{Berlyn GP, Miksche JP. 1976.} Botanical microtechnique and
cytochemistry. Ames, USA: Iowa State University Press.

\textbf{Björklund S, Antti H, Uddestrand I, Moritz T, Sundberg B.
2007.}Cross\selectlanguage{english}-talk between gibberellin and auxin in development of Populus
wood: gibberellin stimulates polar auxin transport and has a common
transcriptome with auxin. \emph{The Plant Journal} \textbf{52:} 499-511.

\textbf{Bollhoner B, Prestele J, Tuominen H. 2012.} Xylem cell death:
emerging understanding of regulation and function.~\emph{Journal of
Experimental Botany} ~\textbf{63:} 1081-1094.

\textbf{Bukatsch F. 1972.} Bemerkungem zur Doppelfarbung
Astra-blau-Safranin. \emph{Mikrokosmos} \textbf{6:} 255.

\textbf{Butto V, Rossi S, Deslauriers A, Morin H. 2019.} Is size an
issue of time? Relationship between the duration of xylem development
and cell traits. \emph{Annals of Botany} \textbf{123:} 1257-1265.

\textbf{Caballe G. 1993.} Liana structure, function and selection: a
comparative study of xylem cylinders of tropical rainforest species in
Africa and America.~\emph{Botanical Journal of the Linnean Society}
~\textbf{113:} 41-60.

\textbf{Caballe G. 1998.} Le port autoportant des lianes tropicales: une
synthese des strategies de croissance. \emph{Canadian Journal of
Botany}\textbf{76:} 1703-1716.

\textbf{Camacho C, Madden T, Ma N, Tao T, Agarwala R, Morgulis A.
2008.}BLAST (r) Command Line Applications User Manual. (National Center
for Biotechnology Information (US), 2008). URL
https://www.ncbi.nlm.nih.gov/books/NBK279690/

\textbf{Cano-Delgado A, Yin Y, Yu C, Vafeados D, Mora-Garcia S, Cheng,
JC, Nam KH, Li J, Chory J. 2004.} BRL1 and BRL3 are novel
brassinosteroid receptors that function in vascular differentiation in
Arabidopsis.~\emph{Development} ~\textbf{131:} 5341-5351.

\textbf{Carlquist S. 1981.} Wood anatomy of Nepenthaceae. \emph{Bulletin
of the Torrey Botanical Club} \textbf{108:} 324-330.

\textbf{Carlquist S. 1985.} Observations on functional wood histology of
vines and lianas: vessel dimorphism, tracheids, vasicentric tracheids,
narrow vessels, and parenchyma. \emph{Aliso} \textbf{11:} 139-157.

\textbf{Carpentier SC, Panis B, Vertommen A, Swennen R, Sergeant K,
Renaut J, Laukens K, Witters E, Samyn B, Devreese, B. 2008.} Proteome
analysis of non\selectlanguage{english}-model plants: a challenging but powerful
approach.~\emph{Mass spectrometry reviews} ~\textbf{27:} 354-377.

\textbf{Cassan-Wang H, Goue N, Saidi MN, Legay S, Sivadon P, Goffner D,
Grima-Pettenati J. 2013} . Identification of novel transcription factors
regulating secondary cell wall formation in Arabidopsis.~\emph{Frontiers
in plant science} \textbf{4:} 189.

\textbf{Choat B, Brodie TW, Cobb AR, Zwieniecki MA, Holbrook NM.
2006.}Direct measurements of intervessel pit membrane hydraulic
resistance in two angiosperm tree species. \emph{American journal of
botany}\textbf{93:} 993-1000.

\textbf{Choat B, Lahr EC, Melcher PJ, Zwieniecki MA, Holbrook NM.
2005.}The spatial pattern of air seeding thresholds in mature sugar
maple trees. \emph{Plant, Cell \& Environment} \textbf{28:} 1082-1089.

\textbf{Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M.
2005.} Blast2GO: a universal tool for annotation, visualization and
analysis in functional genomics research.~\emph{Bioinformatics}
\textbf{~21:} 3674-3676.

\textbf{Darwin C. 1875.} \emph{The movements and habits of climbing
plants} . London, UK: J. Murray.

\textbf{Du J, Gerttula S, Li Z, Zhao ST, Liu YL, Liu Y, Lu MZ, Groover
AT. 2020.} Brassinosteroid regulation of wood formation in poplar.
\emph{New Phytologist}\textbf{225:} 1516-1530.

\textbf{Evert RF. 2006.} \emph{Esau's plant anatomy: meristems, cells,
and tissues of the plant body: their structure, function, and
development} . New Jersey, USA: John Wiley \& Sons.

\textbf{Ewers FW, Ewers JM, Jacobsen AL, Lopez-Portillo J. 2007.} Vessel
redundancy: modeling safety in numbers. \emph{IAWA Journal}
\textbf{28:}373-388.

\textbf{Ewers FW, Fisher JB. 1991.} Why vines have narrow stems:
histological

trends in \emph{Bauhinia} (Fabaceae). \emph{Oecologia}\textbf{88}
:233--237.

\textbf{Ewers FW, Fisher JB, Chuis TS. 1989.} Water transport in the
liana \emph{Bauhinia fassoglensis} (Fabaceae). \emph{Plant
Physiology}\textbf{91} :1625--1630.

\textbf{Ewers FW, Fisher JB, Chiu ST. 1990.} A survey of vessel
dimensions in stems of tropical lianas and other growth
forms.~\emph{Oecologia} ~\textbf{84:} 544-552.

\textbf{Fisher J, Ewers F. 1995.} Vessel dimensions in liana and tree
species of \emph{Gnetum}(Gnetales). \emph{American Journal of Botany}
\textbf{82:} 1350--1357.

\textbf{Froelich DR, Mullendore DL, Jensen KH, Ross-Elliott TJ, Anstead
JA, Thompson GA, Pelissier HC, Knoblauch M. 2011.} Phloem ultrastructure
and pressure flow: Sieve-Element-Occlusion-Related agglomerations do not
affect translocation. \emph{Plant Cell} \textbf{23:} 4428-4445.

\textbf{Furuta KM, Yadav SR, Lehesranta S, Belevich I, Miyashima S, Heo
JO, Vaten A, Lindgren O, De Rybel B, Van Isterdael G et al. 2014.} Plant
development. Arabidopsis NAC45/86 direct sieve element morphogenesis
culminating in enucleation. \emph{Science}
\textbf{345:}933--937\textbf{.}

\textbf{Gartner BL. 1991.} Structural stability and architecture of
vines vs. shrubs of poison oak, Toxicodendron
diversilobum.\emph{Ecology} \textbf{72:} 2005-2015.

\textbf{Gasson P, Dobbins DR. 1991.} Wood anatomy of the Bignoniaceae,
with a comparison of trees and lianas. \emph{IAWA Bulletin}
\textbf{12:}389--417.

\textbf{Gentry AH. 1986.} Species richness and floristic composition of
Choco region plant communities. \emph{Caldasia} \textbf{15:} 71-91.

\textbf{Gerolamo CS, Angyalossy V. 2017.} Wood anatomy and conductivity
in lianas, shrubs and trees of Bignoniaceae.~\emph{IAWA Journal}
~\textbf{38:} 412-432.

\textbf{Goda H, Sawa S, Asami T, Fujioka S, Shimada Y, Yoshida S.
2004.}Comprehensive comparison of auxin-regulated and
brassinosteroid-regulated genes in Arabidopsis. \emph{Plant
physiology}\textbf{134:} 1555-1573.

\textbf{Groover AT. 2005.} What genes make a tree a tree?~\emph{Trends
in plant science} ~\textbf{10:} 210-214.

\textbf{Guan P, Ripoll JJ, Wang R, Vuong L, Bailey-Steinitz LJ, Ye D,
Crawford NM. 2017.} Interacting TCP and NLP transcription factors
control plant responses to nitrate availability. \emph{Proceedings of
the National Academy of Sciences} \textbf{114:} 2419-2424.

\textbf{Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden
J, Couger MB, Eccles D, Li B, Lieber M \emph{et al.} 2013.} De novo
transcript sequence reconstruction from RNA-seq using the Trinity
platform for reference generation and analysis.~\emph{Nature protocols}
\textbf{~8:} 1494.

\textbf{Hacke UG, Sperry JS, Wheeler JK, Castro L. 2006.} Scaling of
angiosperm xylem structure with safety and efficiency. \emph{Tree
physiology} \textbf{26:} 689-701.

\textbf{Hirakawa Y, Bowman JL. 2015.} A role of TDIF peptide signaling
in vascular cell differentiation is conserved among euphyllophytes.
Frontiers in plant science \textbf{6:} 1048\textbf{.}

\textbf{Hirakawa Y, Kondo Y, Fukuda H. 2010.} TDIF peptide signaling
regulates vascular stem cell proliferation via the WOX4 homeobox gene in
Arabidopsis.~\emph{The Plant Cell} \textbf{~22:} 2618-2629.

\textbf{Hoth S, Ikeda Y, Morgante M, Wang X, Zuo J, Hanafey MK,
Gaasterland T, Tingey SV, Chua NH. 2003.} Monitoring genome-wide changes
in gene expression in response to endogenous cytokinin reveals targets
in Arabidopsis thaliana. \emph{FEBS Letters} \textbf{554:} 373-380.

\textbf{Hu R, Qi G, Kong Y, Kong D, Gao Q, Zhou G. 2010.} Comprehensive
analysis of NAC domain transcription factor gene family in Populus
trichocarpa. \emph{BMC plant biology} \textbf{10:} 145.

\textbf{Huber W, Carey VJ, Gentleman R, Anders S, Carlson M, Carvalho
BS, Bravo HC, Davis S, Gatto L, Girke T \emph{et al}.
2015.}Orchestrating high-throughput genomic analysis with
Bioconductor.~\emph{Nature methods} \textbf{~12:} 115.

\textbf{IAWA Committee. 1989.} IAWA List of microscopic features for
hardwood identification. IAWA Bull. n.s. \textbf{10:} 219--332.

\textbf{Isnard S, Silk WK. 2009.} Moving with climbing plants from
Charles Darwin's time into the 21st century. \emph{American Journal of
Botany} ~\textbf{96:} 1205-1221.

\textbf{Janssen TA, Holtta T, Fleischer K, Naudts K, Dolman H.
2020.}Wood allocation trade\selectlanguage{english}-offs between fiber wall, fiber lumen, and
axial parenchyma drive drought resistance in neotropical trees.
\emph{Plant, cell \& environment} \textbf{43:} 965-980.

\textbf{Johnson D, Eckart P, Alsamadisi N, Noble H, Martin C, Spicer R.
2018.} Polar auxin transport is implicated in vessel differentiation and
spatial patterning during secondary growth in Populus. \emph{American
Journal of Botany} \textbf{105:} 186-196.

\textbf{Kans J. 2018.} Entrez direct: E-utilities on the UNIX command
line. In Entrez Programming Utilities Help
{[}Internet{]}.~\emph{National Center for Biotechnology Information
(US). https://www. ncbi. nlm. nih. gov/books/NBK179288/. Published} .

\textbf{Kim D, Langmead B, Salzberg SL. 2015.} HISAT: a fast spliced
aligner with low memory requirements.~\emph{Nature methods}
\textbf{~12:}357.

\textbf{Ko JH, Han KH, Park S, Yang J. 2004.} Whole-transcriptome
profiling revealed plant body weight-induced secondary growth in
Arabidopsis and its transcription phenotype.~\emph{Plant Physiology}
\textbf{~135:} 1069-1083.

\textbf{Koops P, Pelser S, Ignatz M, Klose C, Marrocco-Selden K, Kretsch
T. 2011} . EDL3 is an F-box protein involved in the regulation of
abscisic acid signalling in \emph{Arabidopsis thaliana} .~\emph{Journal
of experimental botany} ~\emph{62} : 5547-5560.

\textbf{Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J,
Mimura T, Fukuda H, Demura T. 2005.} Transcription switches for
protoxylem and metaxylem vessel formation. \emph{Genes \& development}
\textbf{19:} 1855-1860.

\textbf{Kwon Y, Kim JH, Nguyen HN, Jikumaru Y, Kamiya Y, Hong SW, Lee H.
2013} . A novel Arabidopsis MYB-like transcription factor, MYBH,
regulates hypocotyl elongation by enhancing auxin
accumulation.~\emph{Journal of experimental botany}
~\textbf{64:}3911-3922.

\textbf{Lee J, Kim H, Park SG, Hwang H, Yoo SI, Bae W, Kim E, Kim J, Lee
H, Heo T \emph{et al}. 2021.}Brassinosteroid\selectlanguage{english}-BZR1/2\selectlanguage{english}-WAT1 module
determines the high level of auxin signalling in vascular cambium during
wood formation. New Phytologist\textbf{230:} 1503-1516.

\textbf{Lens F, Sperry JS, Christman MA, Choat B, Rabaey D, Jansen S.
2011.} Testing hypotheses that link wood anatomy to cavitation
resistance and hydraulic conductivity in the genus \emph{Acer. New
Phytologist} \textbf{190} :709--723.

\textbf{Li S, Lin YJ, Wang P, Zhang B, Li M, Chen S, Shi R,
Tunlaya-Anukit S, Liu X, Wang Z \emph{et al}. 2019.} The AREB1
transcription factor influences histone acetylation to regulate drought
responses and tolerance in \emph{Populus trichocarpa} . \emph{Plant
Cell}\textbf{31:} 663--686.

\textbf{Li L, Xu J, Xu ZH, Xue HW. 2005.} Brassinosteroids stimulate
plant tropisms through modulation of polar auxin transport in Brassica
and Arabidopsis. \emph{The Plant Cell} \textbf{17:} 2738-2753.

\textbf{Liu C, Yu H, Rao X, Dixon R. 2021.} Absciscic acid regulated
secondary cell-wall formation and lignin deposition in \emph{Arabidopsis
thaliana}through phosphorylation of NST1. \emph{Proceedings of the
National Academy of Sciences} \textbf{118:} e2010911118.

\textbf{Lohmann LG, Taylor CM. 2014.} A new generic classification of
tribe Bignonieae (Bignoniaceae) 1\textbf{.} \emph{Annals of the Missouri
Botanical Garden} \textbf{99:} 348-489.

\textbf{Lorenzo O, Piqueras R, Sanchez-Serrano JJ, Solano R.
2003.}ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and
jasmonate pathways in plant defense. \emph{Plant Cell} 15: 165--178.

\textbf{Lu D, Wang T, Persson S, Mueller-Roeber B, Schippers JH.
2014.}Transcriptional control of ROS homeostasis by KUODA1 regulates
cell expansion during leaf development. \emph{Nature
communications}\textbf{5:} 3767.

\textbf{MacMillan CP, Mansfield SD, Stachurski ZH, Evans R, Southerton
SG. 2010.} Fasciclin\selectlanguage{english}-like arabinogalactan proteins: specialization for
stem biomechanics and cell wall architecture in Arabidopsis and
Eucalyptus.~\emph{The Plant Journal} \textbf{~62:} 689-703.

\textbf{Makila R, Wybouw B, Smetana O, Vainio L, Sole-Gil A, Lyu M, Ye
L, Wang X, Siligato R, Jenness MK \emph{et al}. 2023.} Gibberellins
promote polar auxin transport to regulate stem cell fate decisions in
cambium. Nature plants\textbf{9:} 631-644.

\textbf{Manni M, Berkeley MR, Seppey M, Simao FA, Zdobnov EM.
2021.}BUSCO update: novel and streamlined workflows along with broader
and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic,
and viral genomes. \emph{Molecular biology and evolution} \textbf{38} :
4647-4654.

\textbf{Mao JL, Miao ZQ, Wang Z, Yu LH, Cai XT, Xiang CB.
2016.}Arabidopsis ERF1 mediates cross-talk between ethylene and auxin
biosynthesis during primary root elongation by regulating ASA1
expression. \emph{PLoS genetics} \textbf{12:} e1005760.

\textbf{Matsumoto-Kitano M, Kusumoto T, Tarkowski P, Kinoshita-Tsujimura
K, Vaclavikova K, Miyawaki K, Kakimoto T. 2008.} Cytokinins are central
regulators of cambial activity.~\emph{Proceedings of the National
Academy of Sciences} ~\textbf{105:} 20027-20031.

\textbf{Mazur E, Benkova E, Friml J. 2016.} Vascular cambium
regeneration and vessel formation in wounded inflorescence stems of
Arabidopsis.~\emph{Scientific reports} ~\textbf{6:} 33754.

\selectlanguage{polish}\textbf{Mazur E, Kurczyńska EU, Friml J. 2014.}\selectlanguage{english} Cellular events during
interfascicular cambium ontogenesis in inflorescence stems of
Arabidopsis.~\emph{Protoplasma} ~\textbf{251:} 1125-1139.

\textbf{Melder E., Bossinger G, Spokevicius AV. 2015.} Overexpression of
ARBORKNOX1 delays the differentiation of induced somatic sector analysis
(ISSA) derived xylem fibre cells in poplar stems. \emph{Tree Genetics \&
Genomes} \textbf{11:} 1-8.

\selectlanguage{ngerman}\textbf{Ménard L, McKey D, Rowe N. 2009.} Developmental plasticity and
biomechanics of treelets and lianas in Manihot aff. uinquepartite
(Euphorbiaceae): a branch-angle climber of French Guiana. \emph{Annals
of Botany} \textbf{103:}1249-1259.

\textbf{Mrad A, Johnson DM, Love DM, Domec JC. 2021.} The roles of
conduit redundancy and connectivity in xylem hydraulic
functions.\emph{New Phytologist} \textbf{231:} 996-1007.

\textbf{Muñiz L, Minguet EG, Singh SK, Pesquet E, Vera-Sirera F,
Moreau-Courtois CL, Carbonell J, Blázquez MA, Tuominen H. 2008.}ACAULIS5
controls Arabidopsis xylem specification through the prevention of
premature cell death. \emph{Development} \textbf{135:} 2573-2582.

\textbf{Nakajima K, Furutani I, Tachimoto H, Matsubara H, Hashimoto T.
2004.} SPIRAL1 encodes a plant-specific microtubule-localized protein
required for directional control of rapidly expanding Arabidopsis
cells.~\emph{The Plant Cell} \textbf{16:} 1178-1190.

\textbf{Nieminen K, Immanen J, Laxell M, Kauppinen L, Tarkowski P,
Dolezal K, Tahtiharju S, Eloa A, Decourteixa M, Ljung K \emph{et al}.
2008.} Cytokinin signaling regulates cambial development in
poplar.~\emph{Proceedings of the National Academy of Sciences}
~\textbf{105:} 20032-20037.

\textbf{Novitskaya LL, Tarelkina TV, Galibina NA, Moshchenskaya YL,
Nikolaeva NN, Nikerova KM, Podgornaya MN, Sofronova IN, Semenova LI.
2020.} The formation of structural abnormalities in Karelian birch wood
is associated with auxin inactivation and disrupted basipetal auxin
transport. \emph{Journal of Plant Growth Regulation}
\textbf{39:}378-394.

\textbf{Obaton M. 1960.} Les Lianes ligneuses à structure anormale des
forêts denses d'Afrique occidentale. \emph{Annales des Sciences
Naturelles Botanique et Biologie Végétale} \textbf{12:} \emph{1--20.}

\textbf{Pace MR, Lohmann LG, Olmstead RG, Angyalossy V. 2015.} Wood
anatomy of major Bignoniaceae clades. \emph{Plant Systematics and
Evolution} \textbf{301:} 967-995.

\textbf{Park MY, Kim SA, Lee SJ, Kim SY. 2013.} ATHB17 is a positive
regulator of abscisic acid response during early seedling
growth.\emph{Molecules and Cells} \textbf{35: 125-133.}

\textbf{Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C.
2017.}Salmon provides fast and bias-aware quantification of transcript
expression.~\emph{Nature methods} ~\textbf{14:} 417.

\textbf{Perry ZD, Saminathan T, Arun A, Vaidya BN, Basu C, Reddy UK,
Joshee N. 2021.} Transcriptome analysis of cambium tissue
of\emph{Paulownia} collected during winter and spring. \emph{Diversity}

\textbf{13: 423.}

\selectlanguage{english}\textbf{Poorter L, McDonald I, Alarcón A, Fichtler E, Licona JC,
Peña-Claros M, Sterck F, Villegas Z, Sass-Klaassen U. 2010.} The
importance of wood traits and hydraulic conductance for the performance
and life history strategies of 42 rainforest tree species.~\emph{New
phytologist} ~\textbf{185:} 481-492.

\textbf{R Development Core Team. 2016.} \emph{R: a language and
environment for statistical computing, v.3.3.2.} Vienna, Austria: R
foundation for Statistical Computing. URL http://www.r-project.org.

\textbf{Rajput KS, Gondaliya AD, Moya R. 2021.} Structure of the
secondary xylem and development of a cambial variant in \emph{Serjania
mexicana} (Sapindaceae). \emph{IAWA Journal} \textbf{43:} 103-115.

\textbf{Richter R, Behringer C, Zourelidou M, Schwechheimer C.
2013.}Convergence of auxin and gibberellin signaling on the regulation
of the GATA transcription factors GNC and GNL in \emph{Arabidopsis
thaliana} .\emph{Proceedings of the National Academy of Sciences}
\textbf{110:}13192-13197.

\textbf{Robinson M, McCarthy D, Chen Y, Smyth GK. 2010.} edgeR:
differential expression analysis of digital gene expression
data.~\emph{Bioinformatics} \textbf{4:} 206-207.

\textbf{Robinson MD, Oshlack A. 2010.} A scaling normalization method
for differential expression analysis of RNA-seq data.~\emph{Genome
biology} ~\textbf{11:} 25.

\textbf{Robinson MD, Smyth GK. 2008.} Small-sample estimation of
negative binomial dispersion, with applications to SAGE
data.\emph{Biostatistics} \textbf{9:} 21--332.

\textbf{Robischon M, Du J, Miura E, Groover AT. 2011.} The Populus class
III HD ZIP, \emph{popREVOLUTA} , influences cambium initiation and
patterning of woody stems.~\emph{Plant physiology}
~\textbf{155:}1214-1225.

\textbf{Rowe NP, Speck T. 1996.} Biomechanical characteristics of the
ontogeny \& growth habit of the tropical liana \emph{Condylocarpon
guianense} (Apocynaceae). \emph{International Journal of Plant
Sciences}\textbf{157:} 406-417.

\textbf{Rowe N, Speck T. 2005.} Plant growth forms: an ecological and
evolutionary perspective. \emph{New phytologist} 166: 61-72.

\textbf{Sachs T. 1981.} The control of the patterned differentiation of
vascular tissues. In \emph{Advances in botanical research} . San Diego,
USA: Academic Press, 151-262.

\textbf{Sachs T. 2000.} Integrating cellular and organismic aspects of
vascular differentiation. \emph{Plant and cell physiology}
\textbf{41:}649-656.

\selectlanguage{english}\textbf{Saez A, Apostolova N, Gonzalez-Guzman M, Gonzalez-Garcia MP,
Nicolas C, Lorenzo O, Rodriguez PL. 2004.} Gain\selectlanguage{english}-of\selectlanguage{english}-function and
loss\selectlanguage{english}-of\selectlanguage{english}-function phenotypes of the protein phosphatase 2C HAB1 reveal
its role as a negative regulator of abscisic acid signalling.~\emph{The
Plant Journal} ~\textbf{37:} 354-369.

\emph{Scarpella} \textbf{E, Marcos D, Friml J, Berleth T. 2006.} Control
of leaf vascular patterning by polar auxin transport.~\emph{Genes \&
development} ~\textbf{20:} 1015-1027.

\textbf{Schenck H. 1893.} Beitrage zur Biologie und Anatomie der Lianen
im Besonderen der in Brasilien einheimischen Arten. In Schimper AFW,
ed.\emph{Beitrage zur Anatomie der Lianen: Botanische Mittheilungen aus
den Tropen} . Jena, Germany: Verlag von Gustav Fisher.

\textbf{Schneider CA, Rasband WS, Eliceiri KW. 2012.} NIH Image to
ImageJ: 25 years of image analysis.~\emph{Nature methods}
~\textbf{9:}671.

\textbf{Scholz A, Klepsch M, Karimi Z, Jansen S. 2013.} How to quantify
conduits in wood?~\emph{Frontiers in Plant Science} ~\textbf{4:} 56.

\textbf{Shi D, Jouannet V, Agusti J, Kaul V, Levitsky V, Sanchez P,
Mironova VV, Greb T. 2021.}Tissue-specific transcriptome profiling of
the Arabidopsis inflorescence stem reveals local cellular signatures.
\emph{The Plant Cell}\textbf{33:} 200-223.

\textbf{Shi D, Lebovka I, Lopez-Salmeron V, Sanchez P, Greb T.
2019.}Bifacial cambium stem cells generate xylem and phloem during
radial plant growth. \emph{Development} \textbf{146:} dev171355.

\textbf{Smyth DR. 2016.} Helical growth in plant organs: mechanisms and
significance.~\emph{Development} ~\textbf{143:} 3272-3282.

\textbf{Snow R. 1935.} Activation of cambial growth by pure
hormones.~\emph{New Phytologist} ~\textbf{34:} 347-360.

\textbf{Soffiatti P, Fort E, Heinz C, Rowe NP. 2022.} Trellis-forming
stems of a tropical liana \emph{Condylocarpon guianense} (Apocynaceae):
A plant-made safety net constructed by simple ``start-stop''
development. \emph{Frontiers in Plant Science} \textbf{13.}

\textbf{Sterky F, Regan S, Karlsson J, Hertzberg M, Rohde A, Holmberg A,
Amini B, Bhalerao R, Larsson M, Villarroel R \emph{et al}. 1998.} Gene
discovery in the wood-forming tissues of poplar: analysis of 5,692
expressed sequence tags. \emph{Proceedings of the National Academy of
Sciences}\textbf{95:} 13330-13335.

\textbf{Suer S, Agusti J, Sanchez P, Schwarz M, Greb T. 2011.} WOX4
imparts auxin responsiveness to cambium cells in Arabidopsis. \emph{The
Plant Cell} \textbf{23} : 3247-3259.

\textbf{Torrey JG, Loomis RS. 1967.} Auxin\selectlanguage{english}-cytokinin control of
secondary vascular tissue formation in isolated roots of
Raphanus.~\emph{American Journal of Botany} ~\textbf{54:} 1098-1106.

\textbf{Tyree MT, Davis SD, Cochard H. 1994.} Biophysical perspectives
of xylem evolution: is there a tradeoff of hydraulic efficiency for
vulnerability to dysfunction?~\emph{IAWA journal} ~15: 335-360.

\textbf{Tyree MT, Zimmermann MH. 2002.} Hydraulic architecture of whole
plants and plant performance. In~\emph{Xylem structure and the ascent of
sap} . New York, USA: Springer-Verlag Berlin Heidelberg: 175-214.

\textbf{Uggla C, Moritz T, Sandberg G, Sundberg B. 1996.} Auxin as a
positional signal in pattern formation in plants.~\emph{Proceedings of
the national academy of sciences} ~\emph{93} : 9282-9286.

\textbf{Van Leene J, Hollunder J, Eeckhout D, Persiau G, Van De Slijke
E, Stals H, Van IsterdaelG, Verkest A, Neirynck S, Buffel Y \emph{et
al}. 2010.} Targeted interactomics reveals a complex core cell cycle
machinery in \emph{Arabidopsis thaliana} . \emph{Molecular systems
biology} \textbf{6:} 397.

\textbf{Vo Phan MS, Keren I, Tran PT, Lapidot M, Citovsky V.
2023.}Arabidopsis LSH10 transcription factor and OTLD1 histone
deubiquitinase interact and transcriptionally regulate the same target
genes.\emph{Communications Biology} \textbf{6:} 58.

\textbf{Wang Z, Gerstein M, Snyder M. 2009.} RNA-Seq: a revolutionary
tool for transcriptomics.~\emph{Nature reviews genetics}
~\textbf{10:}57.

\textbf{Wang C, Liu N, Geng Z, Ji M, Wang S, Zhuang Y, Chai G.
2022.}Integrated transcriptome and proteome analysis reveals
brassinosteroid-mediated regulation of cambium initiation and patterning
in woody stem. \emph{Horticulture research} \textbf{9.}

\textbf{Wason J, Bouda M, Lee EF, McElrone AJ, Phillips RJ, Shackel KA,
Matthews MA, Brodersen C. 2021.} Xylem network connectivity and embolism
spread in grapevine (Vitis vinifera L.). \emph{Plant
Physiology}\textbf{186:} 373-387.

\textbf{Wheeler JK, Sperry JS, Hacke UG, Hoang N. 2005.} Inter\selectlanguage{english}-vessel
pitting and cavitation in woody Rosaceae and other vesselled plants: a
basis for a safety versus efficiency trade\selectlanguage{english}-off in xylem
transport.\emph{Plant, Cell \& Environment} \textbf{28:} 800-812.

\textbf{Wilcox H. 1962.} Cambial growth characteristics. In Kozlowski
TT, ed. \emph{Tree Growth} . New York, Ronald Press Co. pp. 57-88.

\textbf{Willige BC, Isono E, Richter R, Zourelidou M, Schwechheimer C.
2011.} Gibberellin regulates PIN-FORMED abundance and is required for
auxin transport-dependent growth and development in Arabidopsis
thaliana. \emph{Plant Cell} \textbf{23:} 2184--2195\textbf{.}

\textbf{Xu P, Kong Y, Song D, Huang C, Li X, Li L. 2014.} Conservation
and functional influence of alternative splicing in wood formation of
Populus and Eucalyptus. \emph{BMC genomics} \textbf{15:} 780.

\textbf{Xie J, Li M, Zeng J, Li X, Zhang D. 2022.} Single\selectlanguage{english}-cell RNA
sequencing profiles of stem\selectlanguage{english}-differentiating xylem in poplar. \emph{Plant
Biotechnology Journal} \textbf{20:} 417.

\textbf{Yamamoto R, Demura T, Fukuda H. 1997.} Brassinosteroids induce
entry into the final stage of tracheary element differentiation in
cultured Zinnia cells. \emph{Plant and Cell Physiology}
~\textbf{38:}980-983.

\textbf{Yang C, Song J, Ferguson AC, Klisch D, Simpson K, Mo R, Taylor
B, Mitsuda N, Wilson ZA. 2017.} Transcription factor MYB26 is key to
spatial specificity in anther secondary thickening
formation.~\emph{Plant physiology} ~\textbf{175:} 333-350.

\textbf{Yang C, Xu Z, Song J, Conner K, Barrena GV, Wilson ZA.
2007.}Arabidopsis MYB26/MALE STERILE35 regulates secondary thickening in
the endothecium and is essential for anther dehiscence.~\emph{The Plant
Cell} ~\textbf{19:} 534-548.

\textbf{Ye ZH, Zhong R. 2015.} Molecular control of wood formation in
trees.~\emph{Journal of experimental botany} ~\textbf{66:} 4119-4131.

\textbf{Yoshida T, Nishimura N, Kitahata N, Kuromori T, Ito T, Asami T,
Shinozaki K, Hirayama T. 2006.} ABA-hypersensitive germination3 encodes
a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid
signaling during germination among Arabidopsis protein phosphatase
2Cs.~\emph{Plant physiology} ~\textbf{140:} 115-126.

\textbf{Yu D, Janz D, Zienkiewicz K, Herrfurth C, Feussner I, Chen S,
Polle A. 2021.} Wood formation under severe drought invokes adjustment
of the hormonal and transcriptional landscape in Poplar.
\emph{International Journal of Molecular Sciences}\textbf{22:} 9899.

\textbf{Zhang J, Eswaran G, Alonso-Serra J, Kucukoglu M, Xiang J, Yang
W, Elo A, Nieminen K, Damen T, Joung J \emph{et al}. 2019} .
Transcriptional regulatory framework for vascular cambium development in
Arabidopsis roots.~\emph{Nature plants} ~\textbf{5:} 1033-1042.

\textbf{Zhang Q, Luo F, Zhong Y, He J, Li L. 2020.} Modulation of NAC
transcription factor NST1 activity by XYLEM NAC DOMAIN1 regulates
secondary cell wall formation in Arabidopsis.~\emph{Journal of
experimental botany} \textbf{71:}1449-1458.

\textbf{Zhbannikov IY, Hunter SS, Foster JA, Settles ML.
2017.}SeqyClean: a pipeline for high-throughput sequence data
preprocessing. In~\emph{Proceedings of the 8th ACM International
Conference on Bioinformatics, Computational Biology, and Health
Informatics:} ~407-416.

\textbf{Zhong R, Demura T, Ye ZH. 2006.} \emph{SND1} , a NAC domain
transcription factor, is a key regulator of secondary wall synthesis in
fibers of Arabidopsis.\emph{The Plant Cell \textbf{18}} \textbf{:}
3158-3170.

\textbf{Zhong R, Kandasamy MK, Ye ZH. 2021.} XND1 regulates secondary
wall deposition in xylem vessels through the inhibition of VND
functions. \emph{Plant and Cell Physiology} \textbf{62:} 53-65.

\textbf{Zhong R, Lee C, McCarthy RL, Reeves CK, Jones EG, Ye ZH.
2011.}Transcriptional activation of secondary wall biosynthesis by rice
and maize NAC and MYB transcription factors. \emph{Plant and Cell
Physiology} \textbf{52:} 1856-1871.

\textbf{Zhong R, Richardson EA, Ye ZH. 2007.} The MYB46 transcription
factor is a direct target of SND1 and regulates secondary wall
biosynthesis in Arabidopsis. \emph{Plant Cell} \textbf{19:} 2776--2792.

\selectlanguage{polish}\textbf{Ziemińska K, Westoby M, Wright IJ. 2015.}\selectlanguage{english} Broad anatomical
variation within a narrow wood density range---a study of twig wood
across 69 Australian angiosperms.~\emph{PLoS One} ~\textbf{10:}
e0124892.

\textbf{Zimmermann MH, Jeje AA. 1981.} Vessel-length distribution in
stems of some American woody plants. \emph{Canadian Journal of
Botany}\textbf{59:} 1882-1892.

\textbf{Zinkgraf M, Gerttula S, Groover A. 2017.} Transcript profiling
of a novel plant meristem, the monocot cambium.~\emph{Journal of
integrative plant biology} ~\emph{59} : 436-449.

\textbf{Supporting Information}

\textbf{Supporting Information Fig. S1.} \emph{B. magnifica} cambium
activity and secondary xylem production assessment.

\textbf{Supporting Information Fig. S2.} Schematic representation
of\emph{B. magnifica} stem segment for RNA sampling.

\textbf{Supporting Information Fig. S3.} RNA-seq experiment workflow.

\textbf{Supporting Information Fig. S4.}\emph{B. magnifica} exclusive
Biological Processes GO terms.

\textbf{Supporting Information Fig. S5.}Expression profiles
multidimensional scaling plot (MDS).

\textbf{Supporting Information Table S1.} \emph{B. magnifica} biological
samples sequencing data, annotated transcripts, associated GO terms, and
read counts.

\textbf{Supporting Information Table S2.} Comparison between GO terms
numbers of \emph{B. magnifica} and model trees \emph{Populus x
euroamericana} and \emph{Eucalyptus grandis}

\textbf{Supporting Information Table S3.} \emph{B.
magnifica}differentially expressed genes (DEGs) and category enrichment
analysis\textbf{.}

\textbf{Supporting Information Table S4.} \emph{B. magnifica} DEGs
hand-curated categories.

\selectlanguage{english}
\FloatBarrier
\end{document}

