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Functional mechanism of bHLH complexes during early
vascular development
Kyoko Ohashi-Ito and Hiroo Fukuda
The vascular system spreads throughout the plant body. This
highly organized network contains several types of cells.
Vascular cell development is initiated during embryogenesis,
and then vascular cells proliferate, form a vascular pattern, and
commit to specific cell fates. Recent molecular genetics and
modeling approaches have increased our understanding of the
molecular mechanisms underlying early vascular development.
Early events during vascular development are tightly linked and
controlled by transcriptional complexes consisting of
LONESOME HIGHWAY (LHW) and TARGET OF
MONOPTEROS5 (TMO5) families. The role of LHW-TMO5 is
tightly coupled with biosynthesis and/or signaling of
phytohormones such as auxin and cytokinin. In this review, we
discuss the regulatory network mediated by LHW-TMO5 during
early vascular development.
Address
Department Biological Sciences, Graduate School of Science, The
University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan
Corresponding authors: Ohashi-Ito, Kyoko (kyoko@bs.s.u-tokyo.ac.jp)
and Fukuda, Hiroo (fukuda@bs.s.u-tokyo.ac.jp)

Current Opinion in Plant Biology 2016, 33:4247

This review comes from a themed issue on Cell signalling and gene
Edited by Kimberley Snowden and Dirk Inze

sieve elements [35]. These studies have identified

NAC-type transcription factors that play central roles in
driving differentiation into different types of vascular cells
[69]. For example, the VND6 NAC-type transcription
factor directly regulates genes involved in programmed cell
death and secondary wall formation through the TERE ciselement that is present in tracheary element-specific promoters [10].
By contrast, the early process of vascular development
was poorly understood. However, recent studies have
identified crucial roles for two families of basic helixloop-helix type (bHLH) transcription factors during early
vascular development: LONESOME HIGHWAY
(LHW) and TARGET OF MONOPTEROS5 (TMO5)
[11,12,13,14,15,16,17]. Each family contains four
members with redundantly overlapping functions. The
LHW family proteins function as heterodimers by interacting with the TMO5 family proteins [11,15]. LHW and
TMO5 family genes are expressed in developing vascular
cells in various organs [15,18]; therefore, the bHLH
complexes may generally regulate early vascular development throughout the plant. This review discusses the
functional role of the LHW family in cooperation with the
TMO5 family during early vascular development in embryos and roots.

Initiation of vascular development

http://dx.doi.org/10.1016/j.pbi.2016.06.003
1369-5266/# 2016 Published by Elsevier Ltd.

Introduction
The plant vascular system begins to develop during embryogenesis. Vascular initial cells are produced at the
center of triangular-stage embryos. These initial cells
subsequently differentiate into several different types of
vascular cells, which coordinately form the vascular network in roots and hypocotyls [1,2]. The vasculature forms
throughout the plant body and performs the crucial functions of conducting water, nutrients, and signals. Primary
vascular development involves vascular cell proliferation,
vascular patterning, commitment of vascular cell fates, and
vascular cell differentiation. A number of studies have
elucidated the gene regulatory networks underlying the
last stage of vascular development, which involves vascular
cell differentiation into tracheary elements, fibers, and
Current Opinion in Plant Biology 2016, 33:4247

Vascular initial cells destined for vascular fates first

emerge in triangular-stage embryos [1,2]. The lower part
of globular-stage embryos consists of three cell layers.
Four cells located in the innermost layer subsequently
divide periclinally to produce a new cell layer containing
four cells. These four newly emerged cells in the center of
the embryo are the vascular initial cells, which subsequently differentiate into all types of vascular cells. The
auxin response regulator MONOPTEROS (MP) has a
key role in this process. Loss-of-function mp mutants fail
to undergo organized periclinal divisions, resulting in very
severe defects in vascular development [19]. Auxin activates MP, and mutants of many auxin-related genes
involved in biosynthesis, transport, and signaling show
severe defects in cell divisions in embryos [2022]. These
results indicate that auxin is indeed essential for cell
divisions in embryos. TMO5 is a direct target of MP
[23]. In tmo5 t5l1 embryos, the four vascular initial cells
are incorrectly formed [15]. lhw embryos also show very
similar defects in the production of vascular initial cells
[12,15]. These results suggest that LHW-TMO5 regulates the production of vascular intial cells. However, how
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bHLH functions during early vascular development Ohashi-Ito and Fukuda 43

LHW-TMO5 regulates the crucial periclinal divisions is

not known so far.

results suggest that local cytokinin biosynthesis contributes to the promotion of vascular cell divisions during
early vascular development.

Vascular cell proliferation

Continuous periclinal cell divisions in the four vascular
initial cells give rise to a number of vascular cells. Loss-offunction mutants of LONELY GUY (LOG) family genes,
which encode cytokinin biosynthetic enzymes, reduce
the number of vascular cells [24,25]. The septuple mutant
of LOG family genes has only a very limited number of
vascular cells. Similarly, the wooden leg mutant, which is
defective in the cytokinin receptor AHK4, also has a
reduced number of vascular cells [26]. This phenotype
also is observed in other cytokinin-response mutants [27
29]. These observations suggest that cytokinin has an
essential role in promotion of vascular cell divisions.
Recent studies demonstrate that LHW and TMO5 control vascular cell proliferation by regulating cytokinin
biosynthesis. Both single lhw mutants and a tmo5 t5l1
double mutant show reduced numbers of vascular cells in
roots, and higher-order mutants of each gene family lead
to highly reduced numbers of vascular cells [11,15,18].
Conversely, overexpression of TMO5, especially simultaneous overexpression of LHW and TMO5 causes excessive
proliferation of vascular cells [13,15]. The resulting
LHW-TMO5 heterodimers upregulate LOG3 and LOG4
expression, which causes active cytokinin production
[13,16]. The cytokinin response occurs in other vascular cells than cells where both LHW and TMO5 are
expressed in a non-cell-autonomous manner [13,16],
because LHW-TMO5 induces ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6)
[12], which suppresses the cytokinin response [30]. These

Vascular patterning
The vasculature pattern is highly organized. In Arabidopsis roots, a row of xylem vessel cells occupies the center,
phloem cells occur in two regions across the xylem row,
and procambial cells are located between the xylem and
the phloem (Figure 1). This organized pattern is established during embryogenesis and maintained in the primary root. The pattern formation accompanies
proliferation of vascular cells. In fact, most of the
above-mentioned mutants that are defective in vascular
cell numbers also display aberrant vascular patterning
[11,15,2529]. This result suggests that cytokinin also
has a role in determining vascular patterning. Further
studies indicate that spatiotemporal cooperation of auxin
and cytokinin is crucial for vascular patterning
[16,31,32]. In heart-stage embryos, two of the four
vascular cells show higher auxin activity due to PINFORMED1 (PIN1)-mediated auxin flow from the two
cotyledon primordia which induces differentiation into
xylem precursor cells [16]. The other two cells, which
have lower auxin activity, likely respond to cytokinin and
then highly divide to form the procambial cell population.
High auxin activity is restricted to xylem precursor cells,
whereas high cytokinin activity is restricted to procambial
cells. This spatial difference of auxin and cytokinin
activities creates and maintains the vasculature pattern.
In addition, there is a mutual inhibition between auxin
and cytokinin signaling [31,32]. In procambial cells, cytokinin regulates the production and localization of PIN

Figure 1

LHW
TMO5

metaphloem

ACL5

companion cell

SACL

procambial cell

ATHB8

metaxylem
protoxylem
pericycle cell

Cytokinin related

protophloem

LOG3/LOG4
AHP6
Cytokinin response
Auxin response
Current Opinion in Plant Biology

Active domains of key factors for vascular development in an Arabidopsis root apical meristem. Cell types are defined as various colors shown in
the left. A line of xylem precursor cells, which includes protoxylem cells (green) and metaxylem cells (light green), is located between procambial
cells (yellow). Phloem cells (orange, pale orange and pink) are localized in two regions along the axis of the xylem cells. Pericycle cells (dark and
light gray) encircle the vascular cells. Expression patterns of key factors and response domains of auxin and cytokinin are shown in the right.
While cytokinin synthesis occurs in the LOG3/LOG4 expressing domain, auxin synthesis domain is unclear.
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Current Opinion in Plant Biology 2016, 33:4247

44 Cell signalling and gene

family proteins, resulting in auxin accumulation in xylem

precursor cells [31]. By contrast, LHW-TMO5 induces
AHP6 in xylem precursor cells, which inhibits the cytokinin response and consequently inhibits cell division
[13,30]. MP is reported to directly regulate AHP6 [31].
As TMO5 also is a target of MP [23], the LHW-TMO5
pathway is also involved in auxin signaling. Therefore, it
is likely that auxin induces AHP6 expression by several
parallel pathways. Recent modeling approaches support
the mutually inhibitory mechanism between auxin and
cytokinin in vascular patterning, and suggest the importance of auxin flux regulation [33]. Auxin transport
requires auxin transporters, whereas cytokinin diffuses
to neighboring cells; differences in transportation versus
diffusion are probably critical for the spatial regulation of
vascular patterning.

Commitment to xylem fate

TMO5 is initially expressed in all four vascular initial cells
in early embryos. Subsequently, TMO5 expression is
restricted to two of the four vascular initial cells, which
develop xylem precursor cells during embryogenesis
[15,16]. The other two vascular initial cells lose
TMO5 expression, appear to undergo cytokinin-mediated
proliferation, and develop procambial cells. In roots, the
expression of TMO5 is restricted to xylem precursor cells
[15]. Therefore, TMO5 may be strongly involved in
determining xylem fate. These observations lead to the
hypothesis that commitment to xylem fate occurs during
a very early stage of vascular development and precedes
commitment to procambial fate during embryogenesis.
Indeed, it is suggested that xylem precursor cells produce
cytokinin, which diffuses to neighboring cells and induces
procambial cell proliferation in early embryos [16]. In
severe cytokinin-response mutants, most vascular cells in
roots differentiate into protoxylem vessels but not procambial cells [2628]. By contrast, LHW is expressed
more widely in early embryos [12,15]. Overexpression
of LHW-TMO5 causes ectopic xylem vessel differentiation in addition to cell divisions [14], suggesting that
TMO5 has a role in commitment to xylem fate.
How does LHW-TMO5 regulate xylem cell fate? Class
III homeodomain leucine zipper (HD-ZIP III) transcription factors may function downstream of LHW-TMO5
signaling. Several studies show that HD-ZIP III proteins
play a positive role in xylem vessel differentiation redundantly and dose-dependently [3436]. Indeed, the HDZIP III gene ATHB8 is regulated by LHW, and its
overproduction can rescue lhw defects in terms of vascular
development [12]. Because ATHB8 also is a direct target
of MP in leaf primordia [37], ATHB8 expression may be
upregulated by two auxin signaling pathways via MP and
LHW-TMO5. Recently, Muller and others reported that
the HD-ZIP III PHABULOSA directly affected auxin
response through MP and IAA20 expression in xylem cell
differentiation [38]. These combined results, together
Current Opinion in Plant Biology 2016, 33:4247

with the fact that auxin biosynthesis is required for

HD-ZIP III-mediated xylem development [39], suggest
that an intricate regulatory network is established by
cooperative auxin signaling, LHW-TMO5 transcriptional
regulation, and HD-ZIP III transcriptional regulation,
which must be a key element of xylem cell fate determination.

Commitment to phloem fate

Commitment to phloem fate also occurs during early
vascular development. The polarly localized membrane
protein OCTOPUS (OPS), which is expressed in vascular
cells in embryos and in phloem initial cells in roots, is
required for protophloem differentiation [40,41]. Therefore, OPS may function during the commitment to phloem fate. In embryos, OPS expression occurs at a later
developmental stage than LHW and TMO5 expression.
This suggests that commitment to phloem fate appears to
occur later than commitment to xylem fate. Recent
studies indicate that OPS positively modulates brassinosteroid signaling by interacting with BIN2 [42], and that a
CLE peptide and receptor (CLE45-BAM3) module is
involved in early phloem development [43]. These findings suggest that intercellular signaling mediated by
brassinosteroids and CLE peptides may be involved in
determining the commitment to phloem fate.
In Arabidopsis, the final differentiation of xylem and
phloem cells such as xylem vessels and sieve elements
does not occur during embryogenesis. Identifying the
mechanism that arrests processes of differentiation in
embryos is an important issue for future research.

Negative feedback is a key regulatory

mechanism that balances vascular
development
ACAULIS5 (ACL5) encodes an enzyme that synthesizes
thermospermine, which is a polyamine that suppresses
xylem differentiation [4449]. A loss-of-function acl5
mutant mimics LHW-TMO5-overexpressing plants in
terms of excessive vascular cell proliferation and xylem
differentiation [17]. The acl5 phenotypes are suppressed
by lhw [14,17]. These genetic results indicate a tight
link between the ACL5 pathway and the LHW-TMO5
pathway. Based on detailed genetic and cell biological
analyses, two research groups proposed a novel regulatory
mechanism in which a negative feedback loop controls
the output levels of LHW-TMO5 [14,17]. In this model, LHW-TMO5 upregulates ACL5 in addition to SUPPRESSOR OF ACAULIS5 1 LIKE (SACL) family genes,
which encode bHLH proteins. The SACL promoter
sequences contain a conserved upstream open reading
frame (uORF) [50], which blocks translation of the main
ORF in the absence of thermospermine [45]. Thermospermine eliminates the suppressive effect of uORF
and enhances SACL production. ACL5 and SACL
proteins suppress the function of LHW-TMO5 through
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bHLH functions during early vascular development Ohashi-Ito and Fukuda 45

Figure 2

hypophysis division delays in lhw embryos [11], and that

TMO5 partially rescues the mp rootless phenotype [23].
These facts imply that LHW-TMO5 plays a leading role
in the root initial formation directly or indirectly via early
vascular development during embryogenesis. In addition
to understanding the functional regulation of LHWTMO5 in vascular development, clarifying the general
function of LHW-TMO5 in the whole plant is a goal for
the future.

HD-ZIP III

Acknowledgements

auxin
?

Proliferation

MP

Commitment
to xylem fate

Patterning
LHW TMO5

LOG3/LOG4

AHP6

cytokinin

ACL5

SACL

Negative feedback
regulation
Current Opinion in Plant Biology

Schematic illustration of key regulators involved in early vascular

development. Transcriptional regulators are shown as boxes and
enzymes are shown as ellipses. LHW and TMO5 function in
transcriptional regulation as a heterodimer. Single-solid and doubleline arrows indicate transcriptional regulation and others, respectively.
SACL indicates SAC51, SACL1, SACL2 and SACL3. Note that SACL
inhibits the function of LHW-TMO5 by making a heterodimer with
LHW. HD-ZIP III includes five genes, but ATHB8 may play the most
important role.

a protein-protein interaction between LHW and SACL.

This negative feedback loop consisting of LHW-TMO5,
ACL5, SACL, and LHW-SACL contributes to regulating
the output levels of LHW-TMO5. Interestingly, previous
work shows that ACL5 is induced by auxin and regulated
by MP [51], and that thermospermine modulates the
expression of auxin-related genes [52]. Therefore, the
input pathways of this negative regulation may be more
complex than currently proposed. As acl5 mutant embryos
do not display an obvious phenotype, this feedback
regulation likely contributes post-embryonically to maintain correct levels of LHW-TMO5.

Research in the authors laboratory on the topic of this manuscript is

supported by Grants-in-Aid from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (25113004 to K.O.-I., and
15H05958 and NC-CARP project to H.F.), from the Japan Society for the
Promotion of Science (15K07098 to K.O.-I. and 23227001 to H.F.) and from
the Naito Foundation to K. O.-I. and H.F.

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Conclusions and perspectives

Transcriptional regulation mediated by LHW-TMO5
plays a central role in all three events of early vascular
development, that is, proliferation, patterning, and commitment to xylem cell fate (Figure 2). Especially, vascular
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proceed by mutual regulation by auxin activity, LHWTMO5, and cytokinin activity. Although details of the
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Current Opinion in Plant Biology 2016, 33:4247