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Physiologia Plantarum 2014 © 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
MINIREVIEW
Auxin is a central player in the hormone cross-talks that
control adventitious rooting
Daniel Ioan Pacurara,†, Irene Perronea,b,†,∗and Catherine Bellinia,c
aDepartment of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, SE-90187, Sweden
bDepartment of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, Umeå, SE-90183,
Sweden
cInstitut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Versailles Cedex, 78026, France
Correspondence
*Corresponding author,
e-mail: Irene.Perrone@slu.se
Received 29 October 2013;
revised 11 February 2014
doi:10.1111/ppl.12171
Vegetative propagation of economically important woody, horticultural and
agricultural species rely on an efficient adventitious root (AR) formation.
The formation of ARs is a complex genetic trait regulated by the interaction
of environmental and endogenous factors among which the phytohormone
auxin plays an essential role. This article summarizes the current knowledge
related to the intricate network through which auxin controls adventitious
rooting. How auxin and recently identified auxin-related compounds affect
AR formation in different plant species is discussed. Particular attention is
addressed to illustrate how auxin has a central role in the hormone cross-talk
leading to AR development. In parallel, we describe the molecular players
involved in the control of auxin homeostasis, transport and signaling, for a
better understanding of the auxin action during adventitious rooting.
Introduction
Unlike the vast majority of species in the animal king-
dom, who reproduce exclusively sexually, asexual repro-
duction, also known as vegetative propagation, vegeta-
tive multiplication or vegetative cloning, is a common
form of reproduction in plants. Although most plants
normally reproduce sexually, for many species vegeta-
tive propagation can occur, either naturally or artificially
when induced by different methods. This capacity of
plants to reproduce vegetatively lays in the extraordi-
nary plasticity of the plant cells to dedifferentiate and
then redifferentiate, forming new organs (e.g. roots or
shoots) that will eventually regenerate a new plant. For a
Abbreviations – 2,4-D, 2,4-dichlorophenoxyacetic acid; ABA, abscisic acid; ABCB1, ATP BINDING CASSETTE TYPE B 1;
AR, adventitious root; ARF, AUXIN RESPONSE FACTOR; ASA1, ANTHRANILATE SYNTHASE ALPHA 1; ASB1, ANTHRANILATE
SYNTHASE BETA 1; BR, brassinosteroids; cGMP, cyclic guanosine monophosphate; CK, cytokinin; CsSCL1, Castanea sativa
SCARECROW-like transcript; ET, ethylene; GBs, gibberellins; IBA, indole-3-butyric acid; IGs, indole glucosinolates; LR, lateral
root; NAA, 1-naphthalene acetic acid; NO, nitric oxide; NPA, naphthylphthalamic acid; PAT, polar auxin transport; QC,
quiescent center; RCE1, RUB-CONJUGATING ENZYME1; rtcs, rootless concerning crown and seminal roots; SA, salicylic acid;
SL, strigolactone; TCL, thin cell layer; TIR1, TRANSPORT INHIBITOR RESISTANT1; WEI2, WEAK ETHYLENE INSENSITIVE 2;
†These authors have equally contributed to the work.
plant segment, usually a stem or a leaf cutting, to be able
to live independently once detached from the mother
plant, the regeneration and development of new roots is
indispensable. These roots initiated post-embryonically
from organs other than roots, are called adventitious
roots (ARs).
AR formation is part of the normal development of
the plant and occurs naturally like in most monocotyle-
donous for which they constitute the main root system,
but can also be induced by stresses such as wound-
ing, flooding, etiolation, or through horticultural prac-
tices used for vegetative propagation of many dicotyle-
donous species (reviewed in Geiss et al. 2009, Oinam
et al. 2011). Artificial vegetative or clonal propagation is
Physiol. Plant. 2014
widely used in horticulture and forestry for multiplication
of elite plants obtained in breeding programs or selected
from natural populations (Hartmann et al. 1990). For
many plants it is a fast and economic method of mul-
tiplication, and for plants with a long generation time, or
poor sexual reproduction, it is the only practically appli-
cable method available. In addition vegetative propaga-
tion is clonal, producing new plants genetically identical
to the mother plant.
In order to overcome the problems associated with the
inability or loss of competence to initiate and develop
AR, and to better understand the entire process, many
studies have been conducted at the physiological, bio-
chemical and molecular levels, both on model and eco-
nomically important species. These studies have shown
that AR formation is a heritable quantitative genetic trait
controlled by multiple endogenous and environmental
factors. Among them, auxin, light, temperature and
mineral nutrition are the most important (reviewed in
Geiss et al. 2009, Li et al. 2009, da Costa et al. 2013).
Auxin has emerged as a central player in stimulating AR
formation, natural auxins and synthetic analogs being
the most powerful exogenous stimulators of AR forma-
tion, used empirically for rooting of cuttings in different
species.
Recent molecular studies conducted on model plant
species confirmed the central role of auxin in AR forma-
tion and unveiled the complex cross-regulatory interac-
tions between auxin and many different phytohormones
in the regulation of this developmental process (Kevers
et al. 2009, Kurepin et al. 2011, Gutierrez et al. 2012).
Auxin acts either synergistically or antagonistically with
other hormones to trigger cascades of events leading to
AR initiation and development. In an attempt to under-
stand better the role of auxin in controlling AR devel-
opment in tea (Camellia sinensis) cuttings, (Wei et al.
2013a) identified 77 transcripts differentially expressed
in single nodal cuttings of C. sinensis treated with or with-
out indole-3-butyric acid (IBA) by suppressive subtractive
hybridization (SSH). The majority of the identified genes
were involved in primary and secondary metabolism and
stress responses, but other functions such as transcription
regulation, cellular transport, transport facilitation, pro-
tein fate and cell cycle regulation were also represented.
Further characterization of these genes is needed for fully
understanding their role in adventitious rooting, but they
definitely provide an additional resource for future stud-
ies.
Beside endogenous factors, environmental stimuli
such as light, wounding and temperature affect this
complex process through the interaction with auxin.
This article does not aim at exhaustively describing
adventitious rooting and its control but focuses on the
recent advances in understanding how concerted action
of auxin with other plant hormones regulates the AR
formation and integrates the molecular players recently
identified in different plant species for being involved in
the control of AR formation.
Anatomy of AR development
The process of AR development consists of three succes-
sive but interdependent physiological phases: (1) induc-
tion (period preceding any histological event), (2) ini-
tiation (cell divisions leading to the formation of inter-
nal root meristems, characterized by the occurrence of
small cells with large nuclei and dense cytoplasm) and
(3) expression phase (development of the typical dome
shape structures, internal growth of root primordia and
root emergence). Depending on the species, there are
two major types of AR formation: (1) in some species of
Salix,Populus or Jasminum preformed ARs initials are
already present in the stem but remain dormant until
stem cuttings are made and placed in conditions favor-
able for rooting. These preformed AR initials likely derive
from cells located in vascular regions. So far their origin
has not been traced back therefore it is difficult to more
specifically determine which cell type is involved. (2) In
other cases, no cells in the intact stem are pre-specified
to form ARs. A supplementary step occurring before
the induction phase, named dedifferentiation, is needed
in this type of rooting, where cells first dedifferentiate
acquiring competence for cell proliferation and organ
regeneration (reviewed in Geiss et al. 2009, Pijut et al.
2011). AR initiate from cells adjacent to or part of the vas-
cular tissues, such as phloem or xylem parenchyma cells,
interfascicular cambium or phloem/cambium junction
(Hartmann and Kester 1983, Jasik and DeKlerk 1997,
Naija et al. 2008, Rigal et al. 2012, Ahkami et al. 2013).
In Arabidopsis ARs initiate from hypocotyl pericycle cells
adjacent to the xylem pole in a similar way as lateral roots
(LRs) do (Boerjan et al. 1995, Della Rovere et al. 2013,
Sukumar et al. 2013), from vascular tissues (cambium
and surrounding tissues) in derooted hypocotyls of older
seedlings in which secondary growth has initiated, or in
stem cuttings (da Rocha Correa et al. 2012, Verstraeten
et al. 2013).
Auxin promotes AR development
in stem cuttings
Exogenously applied auxins have a predictable and con-
sistent effect across plant species in inducing adventi-
tious rooting. In horticultural and agricultural practices,
among the available auxins, IBA is generally the most
commonly used rooting hormone because of its higher
Physiol. Plant. 2014
root-inducing capacity compared with indole 3-acetic
acid (IAA), feature partially due to its greater stability
to light than IAA. After IAA, IBA is the most abundant
natural auxin. Whether it has a biological activity on its
own is an issue, which is not completely addressed and
still is a matter of debate. Indeed, in many species it has
been demonstrated that IBA is converted into IAA and
therefore proposed to be active only after its conversion
(reviewed in Kurepin et al. 2011). Interestingly, IBA is not
degraded or converted to IAA during its long-distance
transport. In addition it was recently shown that IAA
transporters such as AUX1, PIN2, PIN7, ABCB1 (ATP
BINDING CASSETTE TYPE B 1) and ABCB19 do not
transport IBA but that ABCG36 and ABCG37 appear
to efflux IBA, but not IAA (Strader and Bartel 2009).
These results suggest that the independent transport sys-
temsmaybeawaytomovetheinactiveprecursorto
its specific site of action (Strader and Bartel 2011), and
point toward new routes to be investigated for better
understanding the differential roles of IAA and IBA in
promoting AR formation. Depending on the species,
the type of explants and the growth conditions, IBA
can be used alone or in combination with other aux-
ins such as 1-naphthalene acetic acid (NAA) or some-
times 2,4-dichlorophenoxyacetic acid (2,4-D) (reviewed
in Oinam et al. 2011). Oinam et al. (2011) summarized
the advances in the use of different types of auxins and
their concentrations for optimal rooting during vegetative
propagation of several ornamental species, both in vitro
and ex vitro conditions. Similar indications are available
for some important hardwood tree species too (Pijut et al.
2011).
Although Arabidopsis is an annual plant and is not
vegetatively propagated, it has been extensively used
in the last decade to unravel the molecular mecha-
nisms controlling AR formation. Different rooting pro-
tocols have been developed that all highlight the role
of auxin as key determinant for AR induction. Thin cell
layers (TCLs) peeled off from the flower stem and com-
prising the epidermis and the underlying cortex layers
can produce ARs in vitro when maintained in continu-
ous darkness on a medium containing the auxin IBA and
low concentrations of the cytokinin (CK) kinetin (Falasca
et al. 2004). These tissues are usually not developing
AR but when placed in vitro with the appropriate hor-
monal balance, i.e. an auxin/CK ratio in favor to auxin,
they will dedifferentiate and reinitiate an AR develop-
ment program. Auxin is also required for AR formation
from flower stem cuttings or detached leaves, but in these
cases CKs are not needed likely because the endogenous
content is sufficient for stimulating cell division (Ludwig-
Müller et al. 2005, Correa et al. 2012, Verstraeten
et al. 2013).
An important factor limiting the clonal propaga-
tion of woody species is the maturation-related loss of
adventitious rooting competence. Mature and juvenile
tissues, even from the same source plant, can have
different responses to auxin treatment (Pijut et al. 2011).
These results could be explained by epigenetic mod-
ifications happening during the maturation process.
Indeed, chestnut (Castanea sativa) cuttings from mature
trees have an increased level of DNA methylation com-
pared with cuttings from juvenile trees (Hasbun et al.
2007). Characterizing the expression and localization
of the Castanea sativa SCARECROW-like transcript
(CsSCL1) in root-forming and in rooting-incompetent
chestnut shoots, (Vielba et al. 2011) recently pro-
vided a better understanding of the complex cross-talks
between auxin and cellular competence-related sig-
naling pathways. CsSCL1 expression is upregulated
by auxin and interestingly in root-forming shoots, but
not in the rooting-incompetent ones, the transcript
expression is specifically located in the cambial zone
and rooting-competent derivative cells. CsSCL1 is a
SCARECROW-like gene isolated from chestnut shoots
(Sanchez et al. 2007); likewise the expression of the
Pinus radiata SHORT-ROOT gene (PrSHR), the putative
ortholog to the Arabidopsis AtSHR gene, was shown to
increase in the cambial region and in rooting competent
cells of hypocotyl cuttings in the first 24 h post-excision
(Sole et al. 2008). Together with PLHETORA (PLTs)
(Rigal et al. 2012, Trupiano et al. 2013) and WOX
(Della Rovere et al. 2013), SHORT-ROOT (SHR) and
SCARECROW (SCR) proteins work in a spatiotemporal
coordinated way to maintain the cells in a meristematic
and/or division-competent state. Auxin strongly affects
SHR and SCR activity. Expression of SCR-LIKE genes
was significantly increased in response to exogenously
applied auxin in root cuttings of two distantly related tree
species (P. r a d i a t a and C. sativa). This increase of expres-
sion level occurred in the first 24 h of the rooting process,
when the meristematic cells reorganized before the start
of cell division and the appearance of AR primordia,
candidating SCR-LIKE genes as molecular players dur-
ing the earliest stages of AR formation (Sanchez et al.
2007). In Arabidopsis, SHR is required to reactivate
post-embryonically the cell divisions in the root apical
meristem, allowing the growth of the main root. In fact,
mutants in the SHR gene show reduced growth of the
primary root. Interestingly they are characterized by the
progressive reduction of PIN auxin efflux carrier proteins
in the root apical tissue that causes a negative impact
on meristem function (Lucas et al. 2011). SHR is also
required for LR development, as shr mutant plants show
significantly less primordia initiation in respect to the
wild-type. As compensatory mechanism for the loss of
Physiol. Plant. 2014
the root apical meristem shr mutants produce anchor
roots, i.e. AR developing at the root– hypocotyl junction.
This activation could be a response to the supply of
nutrients coming from the aerial parts of the plant that
are in excess for a non-growing primary root. SHR would
not be necessary for AR initiation, like in the case of
the LRs, but instead required for the maintenance of the
indeterminate growth of AR roots since in the shr mutant
they stop growing when they reach the same length as
the primary root (Lucas et al. 2011)
Cuttings can also develop ARs spontaneously without
exogenous auxin supply, as in the case for example of
Pisum sativum and Populus spp. (Nordström and Eliasson
1991, Rigal et al. 2012). However, the formation of these
new roots is still auxin dependent, since the removal of
the shoot apex that causes a reduction of endogenous
auxin content leading to a reduction of the AR number. In
P.sativum and Helianthus annuus it has been shown that
the AR number could be partially restored by exogenous
auxin application (Liu and Reid 1992, Kurepin et al.
2011).
Auxin-related compounds promoting
AR formation
Recently, the effect of different auxins or auxin-related
compounds was investigated on Arabidopsis flower stem
segments (Verstraeten et al. 2013). Whereas IAA, IBA
and NAA induced AR formation, same concentrations
of 2,4-D and picloram promoted only the formation of
callus. Picloram and 2,4-D are also commonly used
as herbicides and are toxic at much lower concentra-
tion than NAA, therefore they are rarely used as rooting
stimulators. NAA showed the strongest rooting activity,
although it has been previously demonstrated to have a
lower binding affinity with the auxin receptor TRANS-
PORT INHIBITOR RESISTANT1 (TIR1) compared with
the natural auxin IAA (Dharmasiri et al. 2005, Kepinski
and Leyser 2005). Authors suggested that observed dif-
ferences among auxins could be the result of different
signal transduction involved, hypothesis that needs to be
demonstrated.
IAA is among the most characterized tryptophan
metabolite, but other tryptophan-derivatives IAA-related
compounds have been reported in plants. Melatonin
was discovered in plants in 1995 and is structurally very
similar to auxin (Dubbels et al. 1995). Recently its role
as new growth regulator for AR formation has emerged.
In 6-day-old derooted Lupinus hypocotyls, melatonin,
as well as IAA, induced the initiation of AR primor-
dia from pericycle cells and affected the number and
length of ARs in a dose-dependent manner. Interestingly
10 𝜇Mof melatonin was more efficient and induced
more and longer roots than 10 𝜇Mof IAA (Arnao and
Hernández-Ruiz 2007). In contrast 10 𝜇Mof melatonin
was inhibitory for adventitious rooting in the commer-
cial sweet cherry rootstocks CAB-6P, Gisela6 and MxM
60, whereas lower concentration (0.5– 1 𝜇M) signifi-
cantly increased the number of roots per explant, and
in some genotypes, the percentage of rooted cuttings
(Sarropoulou et al. 2012). This promoting effect was
potentiated when melatonin was combined with auxin,
the strongest effect being observed in combination with
IBA (Sarropoulou et al. 2012).
Like melatonin, serotonin is a tryptophan derivative
that shares structural similarity with auxin. Recent work
in Arabidopsis suggested that serotonin could act as an
endogenous auxin antagonist and thereby regulate auxin
action during LR and AR formation (Pelagio-Flores et al.
2011). The treatment of seedlings with serotonin caused
a decrease in the expression of auxin-inducible gene
markers in developing lateral and AR primordia and
antagonized the effects of NAA on the expression of the
auxin-inducible genes. Moreover, in contrast to auxin, it
seems that serotonin has no effect on the degradation of
Aux/IAA proteins. The relationship between melatonin,
serotonin and auxin is still obscure but their common
biosynthetic pathway suggests a possible coordinated
regulation. How melatonin and serotonin interact with
auxin is not well understood, but the available results
open new routes for the discovery of new compounds
that might act either synergistically with auxin or circum-
vent the recalcitrance to auxin action.
The modulation of auxin homeostasis
affects AR formation
In Arabidopsis mutations in the SUPERROOT 1 (SUR1)
and SUR2 genes (Table 1), which are involved
in the biosynthesis of indole glucosinolates (IGs)
cause IAA overproduction due to the redirection of
indole-3-acetaldoxime (IAOx), the common intermedi-
ate in IGs and IAA biosynthesis, toward IAA biosynthesis
(Mikkelsen et al. 2004). Arabidopsis superroot mutants
sur1 and sur2 (Boerjan et al. 1995, Delarue et al. 1998)
and the dominant activation-tagged yucca1 mutant,
which overexpresses the flavin monooxygenase-like
YUCCA (Zhao et al. 2001), spontaneously produce
AR on the hypocotyls of light grown seedlings, as a
consequence of auxin overproduction (Table 1). Simi-
larly, rice plants overexpressing OsYUCCA1 gene show
an increase in crown root formation (Yamamoto et al.
2007). In contrast downregulation of IAA biosynthesis
leads to a reduced number of ARs. Indeed mutations
in the ANTHRANILATE SYNTHASE ALPHA 1/WEAK
ETHYLENE INSENSITIVE 2 (ASA1/WEI2) and/or the
Physiol. Plant. 2014
ANTHRANILATE SYNTHASE BETA 1/WEAK ETHYLENE
INSENSITIVE 7 (ASB1/WEI7) were able to suppress the
high-auxin-related AR phenotype of the sur1 and sur2
mutants (Stepanova et al. 2005, Pacurar et al. 2014)
(Table 1).
Differences between easy-to-root and difficult-to-root
genotypes have also been attributed to differences in
the ability to conjugate auxins to their inactive forms.
For example, an easy-to-root cultivar of sweet cherry
(Prunus avium) did not conjugate IBA as rapidly as a
difficult-to-root cultivar. In addition IBA only accumu-
lated in cuttings of the easy-to-root cultivar during several
days after severance, which led the authors to suggest
that the difficult-to-root cultivar was not able to properly
hydrolyze IBA conjugates during the appropriate steps
of AR formation. Interestingly AR formation could be
induced in the difficult-to-root cultivar by application of
an inhibitor of conjugation, confirming the importance
of conjugation mechanisms in the maintenance of auxin
homeostasis and as a key step in AR formation (Epstein
et al. 1993).
Auxin transport a key factor
for AR formation
Polar auxin transport (PAT) through auxin influx and
efflux carriers is an important factor in the adventi-
tious rooting process, as shown in gene expression stud-
ies in derooted pine seedlings (Brinker et al. 2004),
intact rice plants (Xu et al. 2005) and carnation cut-
tings (Oliveros-Valenzuela et al. 2008). Recently, Li
et al. (2012) demonstrated that a coordinated expres-
sion between auxin efflux and influx carriers at the
rooting site is important for AR formation in cotyle-
don segments derived from mango (Mangifera indica L.)
zygotic embryos. The expression of MiPIN1 and four
MiAUX genes increased 4days after the rooting induc-
tion. Whereas MiPIN1 localizes at the distal surface of
the cotyledon segment in respect to the embryo axis, the
expression of MiAUXs genes is localized preferentially
in the proximal cut, suggesting a precise PAT during AR
formation from the distal to the proximal cut surface. In
consequence, ARs develop only at the proximal cut sur-
face where auxin is transported (Li et al. 2012). The use of
naphthylphthalamic acid (NPA), another auxin transport
blocker, has been useful to assess the contribution of PAT
on AR formation in shoot tip cuttings of Petunia hybrida
(Ahkami et al. 2013). Twenty-four hours post-excision,
control cuttings show an auxin accumulation in the root-
ing zone due to the contribution of PAT. The applica-
tion of NPA prevented a 24h peak of IAA, as well as
the increase in activities of sucrose degrading enzymes
essential to establish, in normal conditions, the new sink
that allows the AR formation. As result of this modified
auxin homeostasis, NPA-treated cuttings produced a low
number of roots and at 14 days post-excision only 21%
of rooted cuttings. Among Arabidopsis mutants affected
in auxin transport, the abcb19, which is altered in the
expression of the auxin efflux transporter ABCB19 gene,
showed a significant reduction in AR initiation on the
hypocotyls of derooted seedlings compared with those
of wild-type seedlings (Sukumar et al. 2013). In con-
trast, an ABCG19 overexpressing line developed more
ARs than the wild-type. In the wild-type Arabidopsis
hypocotyls, the excision of the root increases the auxin
flux from the apex to the base by more than fourfold,
leading to increased AR formation. This process was
blocked by the removal of the shoot apex as auxin
source, confirming that the auxin produced in the apex
is required for AR formation. ABCB19 is expressed in
epidermal, vascular and pericycle hypocotyl cells, its
expression increases and the protein accumulates after
root excision. This leads to IAA accumulation at the base
of the hypocotyl driving the formation of AR (Sukumar
et al. 2013).
Mutations in the rice gene orthologous to GNOM1,
crown root-less 4 (crl4) (Kitomi et al. 2008) and Osg-
nom1 (Liu et al. 2009), inhibit crown roots and LRs
(Liu et al. 2009) (Table 1). In Arabidopsis, GNOM1 is a
membrane-associated guanine nucleotide exchange fac-
tor of the ADP-ribosylation factor G protein (ARF-GEF)
required for the intracellular trafficking of the auxin efflux
carrier PIN1 (Steinmann et al. 1999). In rice, the expres-
sion of OsPIN2, OsPIN5b and OsPIN9 is altered in Osg-
nom1 mutant, indicating that PAT involving OsPIN auxin
carriers and regulated by CRL4/OsGNOM1 is required
for crown root and LR initiation.
Once AR primordia are formed, the establishment and
maintenance of the auxin maxima in the quiescent center
(QC) is fundamental to allow their indeterminate growth.
The contribution of the biosynthesis and transport of
auxin in this process is well known in primary and LR
(Benkova et al. 2003) and recently Della Rovere et al.
(2013) investigated the role of auxin in the establishment
of the QC in the Arabidopsis AR tips. Regardless if ARs
are formed in planta from hypocotyl pericycle or from
stem endodermis in in vitro TCLs, the positioning of the
QC depends on the coordination between auxin trans-
port and biosynthesis and CK activity. PIN1 and LAX3
auxin transporters, whose expression is regulated by CK,
determine an auxin maximum at the primordium tip,
where the expression of WOX5 is restricted, positioning
the QC in a similar way as it happens in LR meristem.
Local auxin synthesis by YUCCA6 is also maintained
and contributes to the apical auxin accumulation (Della
Rovere et al. 2013).
Physiol. Plant. 2014
Table 1 . Mutants and transgenic lines affected in adventitious roots development.
Line Gene description Species AR – related phenotype References
sur1 C-S lyase involved in
glucosinolates
biosynthesis
Arabidopsis thaliana +Spontaneous production
of ARs on the
hypocotyls of light
grown seedlings
Boerjan et al. (1995)
sur2 Delarue et al. (1998)
Dominant
activation-tagged
yucca1 mutant
Flavin
monooxygenase-like
enzyme involved in
auxin biosynthesis
A. thaliana +Zhao et al. (2001)
OsYUCCA1-OX Oryza sativa +Increased crown root
formation
Yamamoto et al.
(2007)
wei2 ANTHRANILATE
SYNTHASE 𝛼
ANTHRANILATE
SYNTHASE 𝛽
Tryptophan biosynthesis
A.thaliana −Mutations in the genes
suppress superroot
phenotype
Stepanova et al.
(2005)
wei7 Pacurar et al. (2014)
PtAIL1-OX AP2/ERF transcription
factors
Populus tremula ×alba P.
tremula ×tremuloides
+Earlier AR emergence;
significant increase in
the average root
number per cutting
Rigal et al. (2012)
PtAIL1 RNAi −Reduced number and
delayed emergence of
ARs
PtaERF003-OX P.tremula ×alba +Increased AR number Trupiano et al. (2013)
shr GRAS transcription factors A.thaliana +Production of anchor
roots at the
root-hypocotyl junction;
ARs characterized by
determinate growth
Lucas et al. (2011)
tin WOX5 loss-of-function
mutant
A.thaliana −Reduced ARs number Gonzali et al. (2005)
wox11 WOX transcription factors O.sativa −Delayed initiation of
crown roots
Zhao et al. (2009)
abcb19 Auxin efflux carriers A.thaliana −Significant reduction in
root excision-induced
AR initiation
Sukumar et al. (2013)
Oscand1 Cullin-associated and
neddylation-dissociated
1
O.sativa −Normal initiation of crown
root primordia;
defective AR
emergence
Wang et al. (2011)
crl1/arl1 AS2/LOB domain gene O.sativa −No crown roots Inukai et al. (2005),
Liu et al. (2005)
crl4/Osgnom1 −Reduced crown root
number
Kitomi et al. (2008),
Liu et al. (2009)
rtcs Zea mays −No shoot-borne roots Taramino et al. (2007)
arf6 Auxin response factors A.thaliana −Reduced AR number
arf8 −
ARF17-OX −Gutierrez et al. (2009)
arf8-ARF17-OX −Further reduction in AR
number in respect to
single mutants
−
arf6-ARF17-OX
ARF6-OX +Increased AR number
ARF8-OX miRNA targeting ARF17 +
miR160a-OX +
miR160c-OX +
Physiol. Plant. 2014
Table 1 . Continued
Line Gene description Species AR – related phenotype References
gh3.3 Auxin and jasmonate
conjugating enzymes
A.thaliana +Increased AR number Gutierrez et al. (2012)
gh3.5 +
gh3.6 +
gh3.3-gh3.6 −Reduced AR number
gh3.3-gh3.5
−
gh3.3-gh3.5-gh3.6 −Further reduction in AR number
in respect to double mutants
rce1 Rub conjugating enzyme A. thaliana −Mutations in the gene suppress
superroot phenotype
Pacurar et al. (2014)
The modulation of the auxin signaling
affects the AR formation
Auxin metabolism and transport together generate
the different levels of hormone that are the base of
auxin-regulated responses in the plant. Auxin signaling
translates the different auxin concentrations in cell
responses. The understanding of the molecular mech-
anisms underlying the regulation of auxin signaling
during AR development improved in the recent
years. CULLIN-ASSOCIATED AND NEDDYLATION-
DISSOCIATED 1 (CAND1) regulates SCFTIR1 assembly.
This complex targets members of the Aux/IAA family of
transcriptional regulators for ubiquitin-mediated prote-
olysis in response to auxin in Arabidopsis (Feng et al.
2004). The rice mutant Oscand1 is defective in the emer-
gence of crown roots, although the initiation of crown
root primordia is normal (Wang et al. 2011) (Table 1).
By means of expression analyses performed on laser
microdissected primordia at different developmental
stages, the authors showed that the marker genes asso-
ciated with the G2/M cell cycle transition are repressed
in the meristem of mature crown root primordia in the
mutant compared with the wild-type. The arrest of this
transition causes the failure of root emergence in the
Oscand1 mutant. Information about the role of auxin
signaling in the crown root initiation in rice emerged
also from the studies on crown rootless 1/adventitious
rootless 1 (crl1/arl1) mutants, which entirely lack crown
roots. These mutants are altered in the expression of
the auxin inducible OsLBD3-2 gene encoding a LAT-
ERAL ORGAN BOUNDARIES domain (LBD) protein
(Inukai et al. 2005, Liu et al. 2005) (Table 1). Simi-
larly, the maize mutant rootless concerning crown and
seminal roots (rtcs), mutated in the ortholog of the
rice gene ARL1/CRL1/OsLBD3-2,isimpairedinthe
initiation of crown and seminal roots (Taramino et al.
2007). The rtcs mutation, on the contrary, does not
affect formation and density of LR, suggesting that in
this case the AR formation is under a specific genetic
control.
At high IAA levels, the complex SCFTIR1 promotes
the degradation of the Aux/IAA proteins that leads to
derepression of AUXIN RESPONSE FACTOR (ARF)tran-
scripts and to the expression of auxin responsive genes
(reviewed in Ljung 2013). Gutierrez et al. (2009) showed
that a subtle balance of three different ARFs controls
AR initiation. While AtARF6 and AtARF8, targets of
miR167, work as positive regulators of adventitious root-
ing, AtARF17, target of miR160, is a repressor. A com-
plex regulatory network maintains their balance; the
three ARFs interact genetically and regulate each other’s
expression by modulating the activity of their corre-
sponding microRNA. Three Gretchen Hagen3 (GH3)
genes act downstream of these ARFs to modulate jas-
monate (JA) homeostasis (Gutierrez et al. 2012). Potential
interactions between auxin homeostasis and light in the
regulation of adventitious rooting have been investigated
in different species such as Eucalyptus and Phaseolus
(reviewed in Geiss et al. 2009). In Arabidopsis the posi-
tive and negative regulators described above could repre-
sent the link between auxin and light signaling pathways.
After transfer to the light, the expression of ARF6 and
ARF8 increases in the vascular tissue of the hypocotyl
whereas ARF17 expression decreases (Gutierrez et al.
2009). Moreover, the light quality has been shown to
be relevant too, as ARF6 and ARF8 have a differential
response in far-red light whereas ARF17 expression is not
related to the different light conditions.
Auxin interacts with other hormones in the
regulation of AR formation
The emerging lines of evidence from studies in differ-
ent systems suggest a complex hormonal crosstalk tak-
ing place during AR formation, and auxin likely inter-
acts with nearly all the phytohormones known to be
involved in the process. Moreover different hormonal
Physiol. Plant. 2014
Fig. 1. Auxin cross-regulatory interactions with other hormones and
signaling molecules in the regulation of AR formation. Auxin has a cen-
tral role in the regulation of AR formation, both in intact plants (A) and in
cuttings and in alternative rooting systems (B). Various phytohormones,
such as ethylene (ET), cytokinin (CK), gibberellin (GA), abscisic acid (ABA),
strigolactone (SL), brassinosteroid (BR) and jasmonate (JA), and signaling
molecules salicylic acid (SA) and nitric oxide (NO), have been shown to
influence adventitious rooting either directly, interacting with each other,
or by interacting with auxin at the biosynthesis, transport or/and signal-
ing level. The color code illustrates the species where the interaction was
shown. The dashed lines represent possible interactions.
crosstalk regulates the successive physiological phases
(i.e. induction, initiation and expression) of AR develop-
ment (reviewed by da Costa et al. 2013). These interac-
tions are difficult to study as they differ depending on the
species, the rooting conditions or whether entire plants,
derooted seedlings, stem cuttings or other alternative sys-
tem are used (Fig. 1A, B).
In intact plants ethylene (ET) was shown to stimulate
AR formation both in tomato and rice. A promoting effect
of ET on the AR formation was reported in deep-water
rice, where ET’s activity is potentially co-stimulated by
gibberellins (GAs) and inhibited by abscisic acid (ABA)
(Steffens et al. 2006). ET was shown to positively regulate
AR formation in flooded tomato (Vidoz et al. 2010), and
in in vitro grown tomato seedlings, likely by modu-
lating auxin transport (Negi et al. 2010). ET’s opposite
effect on the AR and LR formation observed in tomato
(Negi et al. 2010) suggests that specificities between
the two developmental processes exist. A promoting
role for ET in AR development has been reported in
cuttings of different species including sunflower, apple,
mung bean or petunia (Geiss et al. 2009, Kurepin et al.
2011) potentially as a result of the auxin-ET crosstalk,
although the mechanisms are not known yet. Although
Zimmerman and Hitchcock (1933) followed by others,
reported the stimulatory role of ET in AR formation,
frequently ET has been shown to have either no effect,
or to act as an inhibitor of rooting (Mudge 1988, De
Klerck et al. 1999). It seems that both ET and CK’s effect
in AR formation is likely to be phase dependent, as both
hormones have a promoting effect in the early induction
phase, while being inhibitory at the late induction phase
(De Klerck et al. 1999, De Klerck 2002, da Costa et al.
2013). The existence of a complex auxin-ET crosstalk
in the control of AR formation was also recently sug-
gested (Pacurar et al. 2014). These authors reported the
use of superroot2-1 (sur2-1) mutant as tool to screen
for suppressors of the AR phenotype (Pacurar et al.
2012, Pacurar et al. 2014). Mutations were identified
in predictable candidate genes involved in either auxin
biosynthesis [ASA1/WEI2,ASB1/WEI7 and TRYPTO-
PHAN SYNTHASE BETA 1 (TSB1)] or signaling [AUXIN
RESPONSE 1 (AXR1),SHORT HYPOCOTYL2/IAA3
(SHY2)andRUB-CONJUGATING ENZYME1 (RCE1)]
confirming the role of auxin as a central regulator of AR
formation. Two of the genes involved in auxin biosyn-
thesis (ASA1/WEI2 and ASB1/WEI7) are also linked to
ET signaling, as the wei2 and wei7 mutants have been
previously described as weak ET insensitive mutants
(Stepanova et al. 2005). Interestingly, the rce1-1 mutant
affected in RCE1, gene required for a proper regulation
of ET biosynthesis (Larsen and Cancel 2004), is also
deficient in auxin and JA response (Dharmasiri et al.
2003). ET probably impacts AR formation by altering
auxin perception, as the suppressor mutated in the RCE1
gene still retains the high IAA content of sur2-1.The
isolation of these mutants as suppressors of sur2 suggests
that an auxin-ET cross-regulatory interaction occurs
during AR formation in Arabidopsis.
Similarly, CKs have been shown to negatively impact
on AR formation in poplar cuttings (Ramirez-Carvajal
et al. 2009) and in rice (Kitomi et al. 2011). Recent
Physiol. Plant. 2014
data suggest that auxin promotes crown root initia-
tion in rice through repression of CK signaling (Kit-
omi et al. 2011). Moreover, exogenous auxin applica-
tions negatively impacted on the local CK biosynthesis
in nodal stems of P.sativum (Tanaka et al. 2006) and
on CK biosynthesis and/or transport in carnation cut-
tings (Agullo-Anton et al. 2014). Nevertheless, CKs were
used in combination with auxin in in vitro conditions to
induce AR formation in microcuttings or TCLs (De Klerck
2002, Falasca et al. 2004, Fattorini et al. 2009). Certain
types of CKs applied at low concentration are required
to enhance rooting in apple microcuttings (De Klerck
2002). Their promoting effect during this early phase is
likely related to the fact that cell divisions require CKs.
GAs have inhibitory effects on AR formation in
poplar cuttings (Busov et al. 2006) and in tomato
(Lombardi-Crestana et al. 2012), while they promote AR
initiation and elongation on deep-water rice (Steffens
et al. 2006). The opposite effects observed might be due
to GAs having different role in AR formation in different
systems, or as for the ET, their function varies during the
distinct phases of the adventitious rooting process.
ABA appears to be a negative regulator of adventitious
rooting, both in tomato and in rice. The ABA-deficient
tomato mutants flacca and notabilis produce an excess
of AR on the stems (Tal 1966, Thompson et al. 2004). In
flooded rice plants, the balance between ET, GA and ABA
is altered upon submergence, and this altered balance
was shown to control various adaptive responses,
including AR formation. ABA was identified as the
hormone that negatively controls AR emergence, which
was reduced to about 50% upon ABA treatment (Steffens
et al. 2006).
Very recently strigolactones (SLs), a new class of phy-
tohormones, were shown to have an inhibitory effect on
AR formation both in Arabidopsis and pea (P.sativum),
as adventitious rooting is enhanced in SL-deficient and
response mutants of both species (Rasmussen et al.
2012b, Rasmussen et al. 2012a). SL might act by modu-
lating the auxin level in the cells or tissues from which the
AR originate, and potentially negatively regulating root-
ward auxin movement (Bennett et al. 2006, Brewer et al.
2009, Crawford et al. 2010, Ruyter-Spira et al. 2011). To
further support this hypothesis, (Rasmussen et al. 2012b)
showed that in Arabidopsis SL can have inhibitory effects
on AR formation, even in the presence of elevated auxin
content.
JA was also shown to play an important role in the reg-
ulation of AR formation in tobacco TCLs and etiolated
Arabidopsis hypocotyls (Fattorini et al. 2009, Gutier-
rez et al. 2012). This is not surprising, as AR formation
is generally stress induced in horticultural practices of
clonal propagation by, e.g. severance (cuttings), changes
in light intensity and/or quality (etiolated stems, layering),
or by temporary hydric stress (flooding). Interestingly,
while transient JA accumulation at the cuttings base in
Petunia is interpreted as critical for the rooting process
by increasing the level of cell wall invertases and sink
strength (Ahkami et al. 2009), or positively modulate
auxin and CK activity in tobacco (Fattorini et al. 2009),
JA was found to be inhibitory for AR formation in Ara-
bidopsis hypocotyl (Gutierrez et al. 2012). In Arabidop-
sis hypocotyls, auxin controls AR initiation by regulat-
ing JA homeostasis and negatively regulating JA signaling
(Gutierrez et al. 2012).
The role of the signaling molecule nitric oxide (NO)
in AR formation and its crosstalk with IAA was inves-
tigated in cucumber (Cucumis sativus). Pagnussat et al.
(2002) first showed that NO mediates the auxin response
during AR formation on hypocotyl cuttings of cucum-
ber, and offered indications that NO acts downstream of
IAA in promoting AR development through the guany-
late cyclase (GC)-catalyzed synthesis of the messen-
ger cyclic guanosine monophosphate (cGMP) (Pagnus-
sat et al. 2003). Subsequently, they suggested that a
MAPK signaling cascade is activated during the adventi-
tious rooting process induced by IAA in a NO-mediated
but cGMP-independent pathway (Pagnussat et al. 2004).
All together these data suggest that, in cucumber cut-
tings, a complex set of cellular messengers, among which
MAPK and cGMP are activated by upstream components
involving IAA and NO, and orchestrate the formation of
a new root system when primary root is removed.
Emerging recent data suggest that another signaling
molecule, salicylic acid (SA), which in plants regulates
disease resistance responses and is involved in various
biotic and abiotic stresses, plays a role in AR formation
too. Arabidopsis mutants altered in salicylic biosynthe-
sis eds5-1 and eds5-2 developed fewer ARs than the
wild-type suggesting a promoting role of SA in AR devel-
opment (Gutierrez et al. 2012). This promoting role was
also observed upon treatment of mung bean (Phaseolus
radiatus L) hypocotyls (Wei et al. 2013b). The authors
have shown that SA significantly improved adventitious
rooting in a dose and time dependent manner and
revealed a distinctive role of SA in rooting via accu-
mulation of free H2O2. In carnation cuttings, SA levels
increased significantly during the determination of root
primordia phase, while exogenous auxin application
dramatically impacted on the SA levels during the early
stages of rooting, suggesting a crosstalk between these
two hormones during adventitious rooting (Agullo-Anton
et al. 2014).
Last, but not least, brassinosteroids (BRs) (Ronsch
et al. 1993), alkamides (Campos-Cuevas et al. 2008,
Mendez-Bravo et al. 2010), polyamines (Rey et al. 1994,
Physiol. Plant. 2014
Heloir et al. 1996) and flavonoids (Curir et al. 1990), have
been reported to play a role in the regulation of adventi-
tious rooting in different species, but their mode of action
still needs to be elucidated.
Concluding remarks
Auxin is with no doubt a master regulator of virtually
all aspects of plant development. It acts at different lev-
els, from basic cellular processes to macroscopic phe-
nomena (Sauer et al. 2013). Auxin exerts its myriad of
functions in a coordinated manner with other growth
regulators such as ET, CK, GA, ABA, SL, BR, SA, JA,
polyamides (PA) and NO, and with environmental stim-
uli such as light, temperature or nutrition. Auxin plays
a central role in AR formation, however, in most of the
published reviews the potential hormonal crosstalk tak-
ing place during AR development is inferred based on
the data available from LR development. A recent review
(Bellini et al. 2014) describes the similarities and differ-
ences between AR and primary and LRs. In this article we
have highlighted the recent knowledge on the specificity
of auxin action in the control of AR development, show-
ing that there is still a long way to go before reaching
a comprehensive understanding of the molecular mech-
anisms underlying auxin action in this developmental
process.
Acknowledgements – We apologize to all colleagues whose
work could not be cited due to space limitation. Research
in our group is supported by the Swedish Foundation for
Strategic Research (SSF), the Swedish Research Council
for Research and Innovation for Sustainable Growth (VIN-
NOVA), the Swedish Research Council (VR), the K. & A.
Wallenberg Foundation and the Carl Trygger Foundation
and the Carl Kempe Foundation.
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