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International Journal of
Molecular Sciences
Review
Understanding the Intricate Web of Phytohormone Signalling
in Modulating Root System Architecture
Manvi Sharma †, Dhriti Singh †, Harshita B. Saksena †, Mohan Sharma †, Archna Tiwari ‡, Prakhar Awasthi ‡,
Halidev Krishna Botta ‡, Brihaspati Narayan Shukla §and Ashverya Laxmi *
Citation: Sharma, M.; Singh, D.;
Saksena, H.B.; Sharma, M.; Tiwari, A.;
Awasthi, P.; Botta, H.K.; Shukla, B.N.;
Laxmi, A. Understanding the
Intricate Web of Phytohormone
Signalling in Modulating Root
System Architecture. Int. J. Mol. Sci.
2021,22, 5508. https://doi.org/
10.3390/ijms22115508
Academic Editor: Andrzej Bajguz
Received: 14 April 2021
Accepted: 13 May 2021
Published: 24 May 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India;
manvi.sharma3@yahoo.com (M.S.); dhritisingh89@gmail.com (D.S.); harshita@nipgr.ac.in (H.B.S.);
sharma.mohan642@gmail.com (M.S.); archnatiwari@nipgr.ac.in (A.T.); prakhar@nipgr.ac.in (P.A.);
bhkrishna@nipgr.ac.in (H.K.B.); bn.shukla07@nipgr.ac.in (B.N.S.)
*Correspondence: ashverya_laxmi@nipgr.ac.in; Tel.: +91-11-26741612 (ext. 180)
† Equal first authors.
‡ Equal second authors.
§ Third author.
Abstract:
Root system architecture (RSA) is an important developmental and agronomic trait that is
regulated by various physical factors such as nutrients, water, microbes, gravity, and soil compaction
as well as hormone-mediated pathways. Phytohormones act as internal mediators between soil and
RSA to influence various events of root development, starting from organogenesis to the formation of
higher order lateral roots (LRs) through diverse mechanisms. Apart from interaction with the external
cues, root development also relies on the complex web of interaction among phytohormones to exhibit
synergistic or antagonistic effects to improve crop performance. However, there are considerable
gaps in understanding the interaction of these hormonal networks during various aspects of root
development. In this review, we elucidate the role of different hormones to modulate a common
phenotypic output, such as RSA in Arabidopsis and crop plants, and discuss future perspectives to
channel vast information on root development to modulate RSA components.
Keywords:
root system architecture; root system plasticity; root development; root tropic responses;
root meristem; phytohormone signalling
1. Introduction
Plants are undeniably the most important source of food, fuel, medicines, and fibres.
Apart from the above ground parts of the plants that are actively involved in photosynthesis,
the hidden half i.e., the root, controls a wide variety of processes such as nutrients and
water acquisition and anchorage in the soil, and also acts as an interface between plant and
the earth atmosphere. By sensing and responding to environmental cues and soil nutrition
conditions, roots are capable of dramatically altering their architecture and hence enable
plants to overcome the challenges posed due to their sessile nature.
Understanding the development and architecture of roots holds potential in improv-
ing crop yield and optimizing agricultural land use. The recent development of newer
technologies such as advanced digital photography, 3D root imaging, transparent soils,
automated rhizotron, X-ray computed tomography, luminescence-fluorescence based imag-
ing systems, and neutron tomography has enabled us to better understand the architecture
of complex root systems [
1
]. Moreover, extensive studies on the Arabidopsis root system
have generated crucial insights on molecular mechanisms regulating root architecture
development. RSA is influenced by the cross-talk of different hormones as well as by
hormone-environment factors that integrate with the plant system via a specific set of
downstream regulators that lead to changes in gene expression, signal transduction, and
metabolic conversions [
2
,
3
]. With the advent of recent technology, the identification of
Int. J. Mol. Sci. 2021,22, 5508. https://doi.org/10.3390/ijms22115508 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2021,22, 5508 2 of 29
crucial downstream regulators and signalling pathways has now become a reality. For
instance, a study by Šimáskováand co-workers has shown that cytokinin (CK) induces
PIN1 and PIN7 expression via CYTOKININ RESPONSE FACTOR 2 (CRF2) and CRF6
in order to modulate RSA. They showed that domains PCRE7 and PCRE1 of PIN1 and
PIN7, respectively, were responsible for their cytokinin-inducibility. A yeast one hybrid
(Y1H) screen, ChIP-qPCR, and a protoplast-based luciferase assay demonstrated that the
CRF2 and CRF6 transcription factors are positive regulators of PIN7 and PIN1 expression.
Additionally, mutant analysis showed that crf236 phenocopies auxin transport defective
mutants, confirming that PINs are targets of these transcription factors [
4
]. Another study
shows that FOUR LIPS (FLP) is a transcriptional regulator of PIN3 in the early stages of
LR development [
5
]. However, such interactions have mainly been characterized in model
plants such as Arabidopsis, and translating this information into economically important
crop plants will open new research possibilities for plant biologists.
This review will briefly outline the root development process from the formation of
an embryonic root to the control of root architecture. It also provides insights on various
aspects of RSA and how they are regulated by various hormones and their crosstalk
in plants.
2. Root System Architecture
The three dimensional structure of the root system is specified as root system ar-
chitecture (RSA). RSA components include the primary root (PR) length, the number,
angle, length, patterning of lateral roots (LR), adventitious roots (AR), and epidermal
cell outgrowth called root hair (RH) [
6
]. There are two types of root systems defined by
their branching patterns and developmental origin: taproots and fibrous roots (
Figure 1
).
Taproots that occur in dicots consist of the PR, RH and secondary smaller LRs, and ARs
(arising from a root-shoot junction or from the hypocotyl) (Figure 1A). In addition to RH
and primary root, a fibrous root system as observed in cereals (Oryza sativa,Zea mays etc.)
consists of dense mass of adventitious roots (AR) that occur along with seminal roots,
crown roots, and LRs (Figure 1B). However, in cereals, the PR dies as the monocots age,
and the adventitious roots form the major portion of the RSA. Depending on the species,
RSA displays high variation in morphological traits that help the plant to adapt to a highly
competitive environment.
2.1. Primary Root Growth and Development
PR is the basic and main component of RSA, is the first to emerge, and is derived
from embryonically formed meristematic tissue that forms the hypophysis near the base
of the early globular embryo. Asymmetric division takes place, giving rise to upper cell
that eventually forms the quiescent centre (QC) and the lower cell that gives rise to the
upper cells of the columella. Numerous stem cells surround the QC and, depending on
their position, give rise to different tissue types such as columella, vascular tissue, ground
tissue, epidermis, and the root cap [7].
The primary root has different developmental zones. In the root apical meristem
(RAM), all cells originate from one or more precursor or stem cells at the very tip, called
stem cell niche (SCN). The daughter cells divide at different proportions and various rates
to generate the division zone (DZ). These cells exit the cell cycle and elongate, generating
the elongation zone (EZ). The cells then differentiate and acquire tissue specific features
based on their radial position. This zone is called the differentiation zone (Diff zone). The
transition zone (TZ) marks the developmental boundary between dividing and differen-
tiating cells [
7
,
8
]. Each developmental zone correlates with the graded distribution of
hormones, mRNA, peptides, TFs, etc. Auxin and CK play an important role in maintaining
the SCN and cell differentiation, respectively. Auxin gradient is expressed as a maximum
in SCN and shows a decline towards the TZ [
9
,
10
]. The expression of PLETHORA (PLT)
TFs correlates with the auxin maxima, declining at the TZ [
11
], and its expression in SCN
is dependent on ARFs such as MONOPTEROS, NON-PHOTOTROPHIC HYPOCOTYL 4
Int. J. Mol. Sci. 2021,22, 5508 3 of 29
(NPH4) [
12
]. In turn, PLT contributes to the establishment of auxin maxima by increasing
auxin biosynthesis and polar auxin transport (PAT). Cell expansion and differentiation
in the root are controlled by CK signalling that promotes the transcription of SHY2 and
GH3.17 genes that further shape the auxin gradient, where they position auxin minima
at the TZ, leading to differentiation. CKs via ARR1 also promote cell wall remodelling
enzyme EXPANSIN1 (EXP1), consequently driving cell differentiation [8].
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 30
Figure 1. (A) Tap root system: RSA of a dicot (tomato) showing embryonic primary root, lateral roots, adventitious roots
of shoot origin (hypocotyl root), and root-shoot junction (junction root). (B) Fibrous root system: RSA of a monocot
(maize) showing embryonic origin primary root and seminal roots, adventitious roots of shoot origin (brace roots) and
root-shoot junction (crown root), laterals, and root tip. (C) Root tip of primary root showing root cap, meristematic zone,
elongation zone, and maturation zone having root hairs.
2.1. Primary Root Growth and Development
PR is the basic and main component of RSA, is the first to emerge, and is derived
from embryonically formed meristematic tissue that forms the hypophysis near the base
of the early globular embryo. Asymmetric division takes place, giving rise to upper cell
that eventually forms the quiescent centre (QC) and the lower cell that gives rise to the
upper cells of the columella. Numerous stem cells surround the QC and, depending on
their position, give rise to different tissue types such as columella, vascular tissue,
ground tissue, epidermis, and the root cap [7].
The primary root has different developmental zones. In the root apical meristem
(RAM), all cells originate from one or more precursor or stem cells at the very tip, called
stem cell niche (SCN). The daughter cells divide at different proportions and various
rates to generate the division zone (DZ). These cells exit the cell cycle and elongate, gen-
erating the elongation zone (EZ). The cells then differentiate and acquire tissue specific
features based on their radial position. This zone is called the differentiation zone (Diff
zone). The transition zone (TZ) marks the developmental boundary between dividing
and differentiating cells [7,8]. Each developmental zone correlates with the graded dis-
tribution of hormones, mRNA, peptides, TFs, etc. Auxin and CK play an important role
in maintaining the SCN and cell differentiation, respectively. Auxin gradient is expressed
as a maximum in SCN and shows a decline towards the TZ [9,10]. The expression of
PLETHORA (PLT) TFs correlates with the auxin maxima, declining at the TZ [11], and its
expression in SCN is dependent on ARFs such as MONOPTEROS,
Figure 1.
(
A
) Tap root system: RSA of a dicot (tomato) showing embryonic primary root, lateral roots, adventitious roots of
shoot origin (hypocotyl root), and root-shoot junction (junction root). (
B
) Fibrous root system: RSA of a monocot (maize)
showing embryonic origin primary root and seminal roots, adventitious roots of shoot origin (brace roots) and root-shoot
junction (crown root), laterals, and root tip. (
C
) Root tip of primary root showing root cap, meristematic zone, elongation
zone, and maturation zone having root hairs.
There are various TFs and genes involved in QC and RAM maintenance. These
include WUSCHEL-LIKE HOMEOBOX 5 (WOX5), CYCLIN D3 (CYCD3), CYCLING DOF
FACTOR 4 (CDF4), SCARECROW (SCR), SHORTROOT (SHR), HAIRY MERISTEM (HAM),
etc. (extensively reviewed in [13]).
There are some substantial differences between dicot and monocot root tissue orga-
nization. The cereal tissues are overall larger and more complex with wide cortical cell
layers, as compared to the narrow ones in dicots. Additionally, the QC population is
drastically larger compared to dicots such as Arabidopsis. Monocots possess polyarch
xylem as compared to the tetrarch xylem of dicots. Further, the number of xylems and
phloems in dicots vary from 2–8 compared to more than 8 in monocots. In dicots, the pith
region is either absent or small and underdeveloped; whereas in monocots the pith is larger
and well-developed [14].
Int. J. Mol. Sci. 2021,22, 5508 4 of 29
2.2. Lateral Root Development
LRs provide better anchorage in soil, help in nutrient assimilation and water uptake,
and make symbiotic association with microbes. LR development is primarily governed by
auxin activity and auxin response maxima; the downstream signalling cascade regulates
LR patterning in angiosperms [
15
–
17
]. LR formation occurs in the single-layered pericycle
tissue at the xylem poles, which are termed as xylem pole pericycle (XPP) cells (extensively
reviewed in [
15
]). After continuous anticlinal division and cell elongation, XPP cells move
from meristem to a zone of auxin maximum. However, this auxin maxima is not regular;
rather, it appears to behave in an oscillation manner, as high auxin peaks are interrupted
by low auxin response [
18
]. The occurrence of the auxin maxima can be seen by DR5:GUS
and DR5:luciferase reporters and can be correlated with LR initiation. In monocots, LR
initiation differs, as LRs emerge from phloem poles rather than from XPP. In addition,
there can be numerous phloem poles (usually 10 or more); LRs initiate in longitudinal
files, with the number of files roughly proportional to the stele diameter [
19
]. Similar
to Arabidopsis, the formation of auxin response maxima is important in LR initiation
in cereals such as maize. DR5::RFP lines have indicated the presence of auxin response
maxima in differentiating xylem cells and in cells surrounding the protophloem vessels [
19
].
Additionally, genome-wide transcriptome analysis of LR initiation has highlighted the
presence of a common transcriptional regulatory pathway between Arabidopsis and maize,
suggesting that genes involved in LR initiation are conserved across angiosperms [20].
2.3. Adventitious Root Development
Postembryonic roots arising from tissues other than roots are termed adventitious.
These may be crown roots (from nodes below the ground), brace root (from nodes above
the ground), stem roots, or seminal roots [
21
]. Different root types contribute to structural
and absorbance functions. Primary and seminal roots are mostly important in the first
stages of plant growth. Brace roots, which take over at later stages of the monocot RSA,
play an important role in the structural support of the plants [22].
2.4. Root Hair Development
RH are tip growing extensions from the root epidermis that not only increase the root
surface area for better nutrient and water acquisition but also facilitate adhesion of the
root to the surrounding rhizosphere and interaction of the root with soil microorganisms
such as arbuscular mycorrhizal fungi and nitrogen-fixing bacteria. Several reports cite that
overall plant fitness is compromised in several loss-of-function RH mutants when grown
in challenging soil conditions [
23
,
24
]. Hence, dynamic RH morphogenesis is a trait that can
be exploited agriculturally to improve water and nutrient acquisition in heterogeneous soil
conditions. RH development starts when a position-dependent signal originates from the
underlying cortical cells. Signal perception in epidermal cells that overlie two cortical cells
triggers a transcription factor cascade that ultimately inhibits the expression of GLABRA2
(GL2), a core non-RH-determining transcription factor. This causes the expression of ROOT
HAIR DEFECTIVE 6 (RHD6) and its homolog RHD6-LIKE1 (RSL1). These proteins induce
the expression of downstream basic helix-loop-helix (bHLH) family targets RSL2,RSL4
and homolog of Lotus japonica ROOTHAIRLESS1 (LjRHL1), LRL3 for root hair growth [
25
];
among them, RSL4 is the key regulator (extensively reviewed in [
26
–
28
]). Several hormonal
cues participate in the development and regulation of RH, and their crosstalk is important
to dynamically regulate RH function in changing soil conditions [27].
3. Impact of Hormones in the Regulation of Different Aspects of RSA
3.1. Primary Root
The long standing link between auxin and root development dates back to the time
of its discovery, when it was named as root forming hormone [
29
], and is indispensable
in regulating almost every major phase of root growth and development [
11
,
17
,
30
,
31
].
Low concentrations of auxin have been shown to stimulate PR growth in Arabidopsis
Int. J. Mol. Sci. 2021,22, 5508 5 of 29
and maize, while higher concentrations inhibited root growth through auxin receptor
TRANSPORT INHIBITOR RESPONSE 1 (TIR1) mediated signalling via an unknown non-
transcriptional mechanism [
32
,
33
]. It can be speculated that free Aux/IAA proteins might
promote root growth, and ubiquitinated Aux/IAA proteins may lead to inhibition of root
growth. Among other signals, auxin is a key instructor of root meristem organogenesis
on which multiple phytohormones and other signalling pathways converge to regulate
root development. In QC, polar auxin transport (PAT) is an important prerequisite to
establish auxin response maxima; however, localized auxin production in the roots also
contributes to the establishment of auxin gradient and maxima required for normal root
development [
34
,
35
]. It was assumed that shoot-derived auxin and its downward transport
contributes to a major portion of auxin in the root to create auxin maxima. However, recent
reports suggest that auxin is produced locally in the roots via a tryptophan-dependent
biosynthesis pathway, which converts tryptophan to indole-3-pyruvic acid (IPyA) to indole-
3-acetic acid (auxin) [
36
,
37
]. WEAK ETHYLENE INSENSITIVE 8 (WEI8) and its homolog
protein TRYPTOPHAN AMINOTRANSFERASE RELATED 2 (TAR2) are the key enzymes
that produce and maintain auxin homeostasis in the roots, thereby establishing the root
meristem. Plants mutated in wei8tar2 displayed loss of root meristem and reduced root
auxin activity. Additionally, through a combination of grafting experiments, Brumos et al.
2018 showed that shoot-derived auxin could not rescue deficiencies of auxin production in
roots [
36
]. Another tryptophan-dependent auxin biosynthesis pathway, which contributes
to a lower but significant amount of auxin in roots, was also reported where the root-
derived auxin biosynthesis was abolished in cyp79B2cyp79B3, thus suggesting multiple
sources of auxin production in roots [
34
]. Localized auxin production and response machin-
ery along with PAT work together to develop flux separation to maintain auxin maxima
in QC [
38
]. There is also a feedback mechanism of WOX5 and INDOLE-3-ACETIC ACID
INDUCIBLE 17 (IAA17) in maintaining auxin maximal response in QC cells that regulate
the root patterning in distal stem cell niche. WOX5 activity induces auxin production in
QC; however, auxin negatively regulates the WOX5 transcription [
39
]. IAA17 causes the in-
hibition of auxin response and therefore promotes WOX5 transcription. IAA17-dependent
auxin response suppression restricts the WOX5 expression in QC and therefore establishes
the identity of root stem cells. Gain-of-function mutant of IAA17(axr3-1) displayed en-
hanced auxin maxima in QC and reduced auxin response in distal stem cells, leading to
the inhibition of root distal stem cell differentiation [38].
In a recent report, a rice gene named OsFPFL4 modulates root growth by affecting
auxin levels. OsFPFL4 overexpressing plants showed shorter primary roots, which were
due to more auxin accumulation and altered expression of auxin biosynthesis and transport-
related genes [
40
]. Another gene encodes for NITRILASE 1 enzyme in Arabidopsis, which
regulates root growth by modulation of auxin biosynthesis. NIT1 overexpressing plants
showed shorter primary roots, which were due to drastic changes of both free IAA and IAN
levels [
41
]. In rice, OsMADS25 regulates root growth through an increased accumulation
of auxin in the root. OsMADS25 promoted auxin biosynthesis as well as transport. At
the same time, it also reduced auxin degradation to stimulate root growth [
42
]. IAA also
induces cyclic guanosine 3
0
,5
0
-monophosphate (cGMP) accumulation in Arabidopsis roots.
Exogenous application of cGMP derivative 8-bromo-cGMP increases auxin dependent
primary root growth. 8-bromo-cGMP mediates this response through the degradation
of AUX/IAA proteins [
43
]. Phosphatidylinositol-specific phospholipase C2 also governs
root growth in Arabidopsis as plc2 mutant showed defects in auxin-mediated multiple
root growth phenotypes, including primary root growth [
44
]. An Arabidopsis exocyst
complex subunit SEC6 gene regulates primary root growth through polar auxin transport
and PIN protein recycling [
45
]. WRINKLED1 (WRI1), a key transcriptional regulator of
fatty acid biosynthesis, regulates primary root growth through alteration in auxin home-
ostasis. The wri1-1 loss-of-function mutants showed an increased amount of indole-3-acetic
acid (IAA)-Asp conjugates and increased abundance of GH3.3, a gene involved in auxin
degradation [
46
]. In rice, auxin influx carrier OsAUX3 regulates root development as
Int. J. Mol. Sci. 2021,22, 5508 6 of 29
Osaux3-1 and Osaux3-2 mutants showed shorter primary roots (PRs) [
47
]. In addition, a
WUSHEL-related homeobox protein, OsWOX4, regulates primary root elongation in rice.
OsWOX4 regulates this response by direct promoter binding and transcriptional activation
of OsAUX1 [
48
]. A multidrug and toxic compound extrusion (MATE) transporter also
governs root development through the modulation of auxin homeostasis in the roots [49].
In soybean, GmYUC2a is an important regulator of auxin biosynthesis during root de-
velopment and nodulation [
50
]. Beyond transcriptional control, auxin may regulate root
growth responses through other, alternate mechanisms such as protein phosphorylation.
Through the impact of different auxin concentrations on the root tip, Nikonorova et al.
2021 identified global auxin-mediated changes in protein abundance and phosphorylation
regulating root growth promotion and inhibition [
51
]. This MS-based phosphoproteome
approach identified novel growth regulators, such as members of the receptor-like kinases
and MAP kinases. This study also suggests that auxin, H+-ATPases, cell wall modifications,
and cell wall-sensing receptor-like kinases are tightly embedded in pathways underlying
cell elongation. In addition, MKK2 was found to be a potential novel regulator of root
growth regulating auxin biosynthesis and signalling [51].
Auxin regulates PR development via interaction with various other phytohormone sig-
nalling components. For example, a plant-specific and ethylene-responsive HOMEOBOX
PROTEIN52 (HB52) mediates crosstalk between auxin and ethylene signalling in control-
ling PR elongation. HB52 works downstream to ETHYLENE-INSENSITIVE3 (EIN3) and
regulates the transcription of auxin transport-related genes, including PIN2,WAVY ROOT
GROWTH1 (WAG1), and WAG2 through binding to their promoter regions (Figure 2A) [
52
].
In another report, ETHYLENE RESPONSE FACTOR1 (ERF1) mediates the interaction of
auxin and ethylene signalling through the transcriptional regulation of ANTHRANILATE
SYNTHASE α1(ASA1) during PR elongation [53].
Numerous studies have shown how the complex interplay between CK and auxin
governs stem cell architecture and meristem size during the early stages of root develop-
ment. The CK transmembrane receptor ARABIDOPSIS HISTIDINE KINASE 3 (AHK3)
together with type-B ARABIDOPSIS RESPONSE REGULATORS (ARR1) and ARR12 mod-
ulate auxin redistribution in the root meristem [
54
]. This change in auxin distribution is
brought by modulating the levels of PINs through activation of the auxin repressor SHORT
HYPOCOTYL 2 (SHY2)/INDOLE-3-ACETIC ACID INDUCIBLE 3 (IAA3), leading to cell
differentiation [
4
,
54
]. CK was also shown to regulate the PIN gene expression transcrip-
tionally through CYTOKININ RESPONSE FACTORS (CRFs) [
4
]. Auxin, on the other hand,
mediates the degradation of SHY2, which leads to the upregulation of PIN genes, leading
to cell division and expansion [
54
,
55
]. Thus, CKs and auxin are involved in the homeostatic
feedback regulatory loop that intersects at the common regulatory factor SHY2 to control
root meristem size [
4
,
56
]. Negative regulators of CK signalling, namely the type-A ARRs,
also influence the patterning of root apical meristem [
57
]. Higher order mutants of type-A
ARRs have been shown to have smaller root meristem sizes as compared to the wild type
and exhibited enhanced sensitivity towards PAT inhibitor NPA. Following exogenous
application of CK, the level of PIN4-GFP was significantly reduced in the root cap. The
protein levels, but not the transcript levels, of auxin efflux carriers PIN1 and PIN3 were
downregulated in the type-A ARR octuple mutant (arr3,4,5,6,7,8,9,15) roots [57].
Int. J. Mol. Sci. 2021,22, 5508 7 of 29
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 30
Figure 2. Regulation of primary root (PR) growth by various hormones and their crosstalk. (A) Auxin effects root growth
in a concentration-dependent manner and interacts with ET signalling pathway to control PR elongation. Low concen-
tration of auxin promotes PR growth probably via free AUX/IAAs, while high concentration inhibited PR growth through
TIR1-mediated signalling via an unknown non-transcriptional mechanism (B) CK signalling modulates PR growth via
AUX-1 mediated auxin translocation. (C) BR induces ET production via stabilizing ACS5, and ACS9 represses ET syn-
thesis through BES1 and BZR1-mediated repression of ACS7, ACS9, and ACS11 to control PR growth. (D) MYC2, a DNA
binding bHLH transcription factor in JA signalling, represses the transcriptional expression of PLT1 and PLT2. JA regu-
lates expression of ASA1 and modulates auxin levels to cause root growth inhibition. JA and auxinsignalling occurs via
AXR1 to control PR growth. (E) SA via NPR1-mediated signalling establishes an auto regulatory feedback regulation
between CK2 and SA to link between SA signalling and auxin transport. SA also led to inhibition of PR elongation in
NPR1-independent manner via affecting PP2A, leading to changes in PIN activity and auxin export, resulting in attenu-
ation of root growth. Solid black arrow indicates confirmed pathway. Blue arrow indicates low levels of auxin, green ar-
row indicates high levels of auxin. Red circle indicates phosphorylation.
Numerous studies have shown how the complex interplay between CK and auxin
governs stem cell architecture and meristem size during the early stages of root devel-
opment. The CK transmembrane receptor ARABIDOPSIS HISTIDINE KINASE 3 (AHK3)
together with type-B ARABIDOPSIS RESPONSE REGULATORS (ARR1) and ARR12
modulate auxin redistribution in the root meristem [54]. This change in auxin distribu-
tion is brought by modulating the levels of PINs through activation of the auxin repressor
SHORT HYPOCOTYL 2 (SHY2)/INDOLE-3-ACETIC ACID INDUCIBLE 3 (IAA3), lead-
ing to cell differentiation [4,54]. CK was also shown to regulate the PIN gene expression
transcriptionally through CYTOKININ RESPONSE FACTORS (CRFs) [4]. Auxin, on the
other hand, mediates the degradation of SHY2, which leads to the upregulation of PIN
genes, leading to cell division and expansion [54,55]. Thus, CKs and auxin are involved in
the homeostatic feedback regulatory loop that intersects at the common regulatory factor
SHY2 to control root meristem size [4,56]. Negative regulators of CK signalling, namely
the type-A ARRs, also influence the patterning of root apical meristem [57]. Higher order
mutants of type-A ARRs have been shown to have smaller root meristem sizes as com-
pared to the wild type and exhibited enhanced sensitivity towards PAT inhibitor NPA.
Following exogenous application of CK, the level of PIN4-GFP was significantly reduced
in the root cap. The protein levels, but not the transcript levels, of auxin efflux carriers
PIN1 and PIN3 were downregulated in the type-A ARR octuple mutant
(arr3,4,5,6,7,8,9,15) roots [57].
ASA1
PIN2, WAG1,
WAG2
PR gro wth
?
Type-B ARRs
Type-A ARRs
Auxin
ACS5,
ACS9
ACS7,
ACS9,
ACS11
Ethylene
PLT1,
PLT2
ASA1 AXR1
Auxin
TIR1
EIN3
HB52
HB52
ERF1
AUX1
BES1
BZR1 MYC2 PP2A
NPR1
PIN P
Auxin
PID/
D6PK/
MPK
CK2
PIN4, PIN7
P
AB C D E
Ub
Ub
Ub
AUX/ IAA AUX/IAAAUX/ IAA
AUX/ IAA
?
CK BR JA SA
Auxin
Figure 2.
Regulation of primary root (PR) growth by various hormones and their crosstalk. (
A
) Auxin effects root growth in
a concentration-dependent manner and interacts with ET signalling pathway to control PR elongation. Low concentration
of auxin promotes PR growth probably via free AUX/IAAs, while high concentration inhibited PR growth through TIR1-
mediated signalling via an unknown non-transcriptional mechanism (
B
) CK signalling modulates PR growth via AUX-1
mediated auxin translocation. (
C
) BR induces ET production via stabilizing ACS5, and ACS9 represses ET synthesis through
BES1 and BZR1-mediated repression of ACS7,ACS9, and ACS11 to control PR growth. (
D
) MYC2, a DNA binding bHLH
transcription factor in JA signalling, represses the transcriptional expression of PLT1 and PLT2. JA regulates expression of
ASA1 and modulates auxin levels to cause root growth inhibition. JA and auxinsignalling occurs via AXR1 to control PR
growth. (
E
) SA via NPR1-mediated signalling establishes an auto regulatory feedback regulation between CK2 and SA to
link between SA signalling and auxin transport. SA also led to inhibition of PR elongation in NPR1-independent manner
via affecting PP2A, leading to changes in PIN activity and auxin export, resulting in attenuation of root growth. Solid black
arrow indicates confirmed pathway. Blue arrow indicates low levels of auxin, green arrow indicates high levels of auxin.
Red circle indicates phosphorylation.
External application of CK or increased CK signalling by mutants (higher order type-A
ARR) shows negative regulation of PR length both in monocots and dicots [
4
,
58
–
63
]. The
negative role of CK in root growth is also supported by CK deficient mutants, where the
PR grows faster [
58
]. However, a study on over-expression of cytoplasmic CYTOKININ
OXIDASE 7 (CKX7) showed conflicting results of reduced root developmental phenotype
from that of other CKXs [
64
]. Type-B ARR higher order mutants of Arabidopsis displayed
increased root length, suggesting a negative role of CK in PR length [
65
–
67
]. In contrast to
this general phenomenon, a recent study showed a novel role of type-B ARRs in positively
regulating PR length in rice [
68
]. Lowering of CK signalling through single and double
mutants of CK receptors can increase root meristem size and activity [
58
,
69
]; however,
CK receptor triple mutant is CK-insensitive and shows decreased root meristem size
and activity [
69
–
71
]. This suggests that a minimum of CK signalling is required for PR
development, and inhibitory levels of CK result in lowering of PR growth. CK also controls
mitotic divisions in the QC directly by repressing the expression of auxin influx carriers
AUXIN RESISTANT 1 (AUX1) and LIKE AUXIN RESISTANT (LAX2) in an ARR1- and
ARR12- dependent manner. CK induces mitotic divisions in the QC as evident by the
loss-of-function higher order type A ARRs [
57
]. Consequently, higher endogenous levels
of CK in the CK oxidase mutants, ckx3 and ckx5, displayed higher divisions in the QC
in comparison to the WT [
72
], further indicating the positive role of CK in QC divisions.
However, the divisions were enhanced in the QC of lax2 roots similarly to CK treated plants,
Int. J. Mol. Sci. 2021,22, 5508 8 of 29
suggesting that CK represses LAX2 expression in the QC. The report also shows that LAX2
is a direct transcriptional target of ARR1. Additionally, the reduction of LAX2 transcript
in response to cytokinin was compromised in ahk2ahk4 and arr1-3arr12-1, suggesting that
cytokinin receptors and type B ARR are necessary for the regulation of LAX2 [
72
]. Studies
also demonstrate how auxin-CK genetic circuits are modulated by common regulatory
genes. AUX1-mediated auxin translocation also regulates CK-dependent root growth
inhibition and elongation at the root tip. AUX1 is activated downstream to type B ARR
and leads to shootward auxin transport, thereby causing an increase in auxin activity
and inhibition of cell elongation at the root tip (Figure 2B) [
73
]. Another report shows
that a rice gene encoding NAC transcription factor 2 (OsNAC2) expressed in the PR
tips, crown roots, and LR primordia, affects auxin and CK signalling genes. OsNAC2
physically interacts with the promoters of auxin inactivating GRETCHEN HAGEN (GH3.6
and GH3.8), AUXIN RESPONSE FACTOR 25 (OsARF25), and CYTOKININ OXIDASE 4
(OsCKX4), thus facilitating the integration of auxin and CK signalling to modulate root
development in rice [74].
Brassinosteroid (BR) is another key player regulating root growth and develop-
ment [
75
,
76
]. BR regulates root growth in a dose-dependent manner; both high and
low doses negatively affect PR growth. Both BR signalling mutants as well as bri1-ems-
suppressor 1 (bes1-D) (gain-of-function) mutants have short roots. Additionally, external BR
treatment negatively affects PR length [
77
–
79
]. Contradictory to these reports, in another
study, low concentrations of BRs have been shown to promote root growth in WT plants
as well as BR-deficient mutants [
80
], although the changes are small [
80
]. BR has been
shown to affect both cell proliferation and cell elongation in a concentration-dependent
manner to modulate root meristem size [
81
]. All these reports indicate that an optimal level
of BR is crucial for root growth and meristem homeostasis. There is a complex cross-talk
of BR signalling, BR catabolism, and auxin synthesis culminating in a pattern formation
of BRASSINAZOLE-RESISTANT 1 (BZR1) in the meristem and elongation zone. It has
been found that low levels of BZR1 are required to maintain the QC, whereas high levels
are required in the elongation zone. It has also been shown that a balance between auxin
signalling and BR signalling is required to maintain normal root growth [
77
]. Additionally,
BR biosynthetic mutant de-etiolated-2-9 (det2-9),having a short root phenotype, showed in-
creased biosynthesis of ethylene and thereby high accumulation of ethylene. This short root
phenotype of det2-9 mutant was partially recovered in mutants det2-9acs9 and det2-9ein3eil1,
which are defective in ethylene synthesis and signalling, respectively. Exogenous BR has
been shown to regulate ethylene biosynthesis in a dose-dependent manner. BR induces
ethylene production via stabilizing ACC SYNTHASE 5 (ACS5) and ACC SYNTHASE 9
(ACS9) (Figure 2C). However, BR signalling represses ethylene synthesis through BES1 and
BZR1 mediated repression of ACC SYNTHASE 7 (ACS7), ACS9, and ACC SYNTHASE
11 (ACS11) (Figure 2C). In addition, increased superoxide anions also contribute to the
short root phenotype of det2-9 mutants. BR regulates superoxide accumulation via the
peroxidase pathway [
82
]. All these studies indicate that BR regulates root growth through
multiple mechanisms. Similarly, BR signalling behaves differently in the epidermal and
stele region. BR signalling promotes stem cell proliferation in the epidermal region; how-
ever, it induces differentiation of stem cells in the stele region. BR signalling induces target
genes in the epidermis particularly related to auxin signalling, but mostly represses genes
in the stele [83].
Although involved in mitigating biotic and abiotic stress responses [
84
–
86
], jasmonic
acid (JA) and salicylic acid (SA) are also involved in regulating various parameters of
root growth and development. The inhibitory effect of JA on plant growth was one of
the first physiological responses reported in the 1980s [
87
,
88
]. The first mutant insensitive
to JA-mediated inhibition of root growth was jasmonate resistant 1 (jar1), which was later
cloned and characterised as JA-Ile synthase [
89
]. Apart from this, various components
of JA signalling also participate in this physiological response. A screen for Arabidopsis
mutants unresponsive to root growth inhibition by a bacterial toxin and the JA homologue,
Int. J. Mol. Sci. 2021,22, 5508 9 of 29
coronatine (COR), revealed coronatine insensitive 1 (coi1-1), a null mutant insensitive to
JAs [
90
]. COI1 is at the interface of JA and ethylene (ET) signalling in mediating root growth
inhibition. Previous reports show that coi1-16 shows unresponsiveness to ET induced root
growth inhibition in light, but not in dark. Furthermore, this response did not require any
other components of JA biosynthesis and signalling such as jar1-1,jasmonate insensitive 1
(jin1), allene oxide synthase (aos), or oxophytodienoate-reductase3 (opr3). Thus, the inhibition
of Arabidopsis root growth to 1-aminocyclopropane-1-carboxylate (ACC) is light, COI1
dependent, but JA independent and occurs due to inhibition in cell elongation [
91
,
92
].
MYC2, a DNA binding bHLH transcription factor in JA signalling, acts as an activator
of JA-induced root growth inhibition [
93
]. Previous reports suggest that MYC2 directly
represses the transcriptional expression of PLT1 and PLT2 (Figure 2D) [
94
]. Moreover, JA
may increase auxin levels by inducing the expression of auxin biosynthetic gene ASA1,
thereby leading to root growth inhibition (Figure 2D) [
95
]. Another example of potential
interaction between JA and auxin signalling occurs via AUXIN RESISTANT 1 (AXR1). In
addition to its role in auxin signalling, axr1-1 shows JA-mediated root growth inhibition
and pathogen susceptibility (Figure 2D) [
96
]. Taken together, the above reports propose
that JA signalling is linked to auxin homeostasis, leading to root growth inhibition. An
interesting report claims that both endogenous and exogenous JA have an important role in
governing the root architecture of Helianthus annuus seedlings. Micromolar concentrations
of JA reduced the PR and LR lengths along with LR number by reducing the cortex cell
length and cell divisions. However, when JA biosynthesis inhibitor Ibuprofen (IBU) was
applied, an increase in cell elongation was observed, thus suggesting that endogenous JA
also affect root phenotype in Helianthus annuus [
97
]. Interestingly, auxins were shown to be
not essential for JA induced root growth inhibition in sunflower [
97
]. Studies have shown
that the auxin-JA resistant double mutant (jar1-1axr1-3) was more resistant than the single
mutants to inhibition of root growth by methyl jasmonate (MeJA). In addition, the level of
resistance between single mutant axr1-3 and double mutant jar1-1axr1-3 was not different
when IAA was applied. Additionally, JA produced its phenotype even in the presence of
an auxin transport inhibitor. Besides, auxin produced its phenotype even when ibuprofen
was applied. Thus, JA appears to act through two pathways: one linked to auxin, and a
second via an auxin independent pathway [
97
]. A recent report highlights the synergistic
role of JA and auxin in stem cell activation and root regeneration. Wound induced JA
causes ETHYLENE RESPONSE FACTOR 109 (ERF109) activation, which further stimulates
CYCLIN D6; 1(CYCD6;1) and ETHYLENE RESPONSE FACTOR 115 (ERF115). They then
modulate RETINOBLASTOMA-RELATED (RBR)-SCARECROW (SCR) module to allow
root tissue regeneration. Upon wounding, auxin accumulation also takes place, which then
activates several regeneration regulators of this pathway [98].
SA, in a concentration-dependent manner, regulates root growth and development
through interaction with the auxin machinery. A report by Zhao and coworkers (2015)
suggests that exogenous application of 50
µ
M of SA promoted root waving along with a
reduction in PR elongation in Arabidopsis. This SA-mediated root waving response was
regulated via NITRIC OXIDE-ASSOCIATED PROTEIN1 (AtNOA1) and NONEXPRESSER
OF PATHOGENESIS-RELATED GENES 1 (NPR1). Atnoa1 mutants displayed a disrupted
root waving phenotype as a result of altered auxin distribution at the root tip, suggesting in-
terplay of SA signalling and auxin transporters through AtNOA1 to modulate root waving
in Arabidopsis [
99
]. Another report shows that exogenous application of 100
µ
M SA inhib-
ited root elongation in Arabidopsis [
100
]. Armengot et al. 2014 revealed that the mutants of
protein kinase CK2, a Ser/Thr kinase accumulated high levels of SA, which further resulted
in the impediment of PR growth (Figure 2E). CONSTITUTIVE PATHOGEN RESPONSE
(CPR) mutants having high constitutive levels of SA exhibited a similar phenotype as the
CK2 loss-of-function mutants. In addition, SA enhanced the transcription of CK2 encoding
genes via the NPR1-mediated signalling, thereby implying an auto regulatory feedback
regulation between CK2 and SA [
101
]. Loss of CK2 activity led to reduction in the SA-
mediated repression of auxin transporter genes including PIN4 and PIN7, thus establishing
Int. J. Mol. Sci. 2021,22, 5508 10 of 29
a link between SA signalling and auxin transport [
101
]. Exogenous application of 3
µ
M
SA inhibits PR elongation and causes shortening of cell length. SA led to auxin redistribu-
tion and a pronounced auxin response at the root tip in a PATHOGENESIS–RELATED 1
(PR1)-independent manner. At concentrations higher than 50
µ
M SA, the growth related
processes in the root were ceased. Lower concentrations of SA promoted the expression of
TRP AMINOTRANSFERASE OF ARABIDOPSIS 1(TAA1) and PIN1 and suppressed PIN2
and PIN7 expression. A high concentration of SA upregulated the expression of TAA1 and
inhibited the expression of PIN efflux transporters [
100
]. Utilizing mathematical modelling
of auxin distribution after SA exposure, Pasternak et al. 2019 hypothesized that root tip
accumulated auxin after treatment with low concentration of SA, whereas high concentra-
tions of SA led to depletion in auxin levels. Accumulation of auxin after the application of
a lower level of SA causes the distal meristem to become enlarged. Endodermal periclinal
divisions were higher in Arabidopsis root treated with a lower concentration of SA as a
result of enhanced auxin levels in the endodermis. However, lesser vascular cell files were
demonstrated in SA-treated roots in comparison to control due to reduced accumulation of
auxin in the vascular cells [
100
]. However, administration of 1.5
µ
M SA under stress-free
conditions increased the total root biomass in Zea mays [
102
]. SA-deficient mutant abnormal
inflorescence meristem1 (aim1) in Oryza sativa exhibits a defect in root growth and showed re-
duced meristem activity. Reduced SA levels in aim1 led to further decrement in the reactive
oxygen species (ROS) levels due to enhanced expression of ROS scavenging-related genes.
The authors hypothesize that AIM1 is involved in SA biosynthesis and represses the ROS
scavenging genes to accumulate ROS for higher root meristem activity via induction of
transcriptional repressors WRKY62 and WRKY76 [
103
]. Recent observations by Tan et al.
2020 concluded that SA led to inhibition of PR elongation in an NPR1-independent manner
(Figure 2E). The reduced PR growth was a result of inhibition of serine/threonine PRO-
TEIN PHOSPHATASE 2A (PP2A) activity by SA, leading to increased phosphorylation of
PIN protein by different kinases such as PINOID (PID)/D6PK/MAPKs, thereby affecting
PIN activity andauxin export, resulting in attenuation of root growth (Figure 2E) [104].
3.2. Lateral Roots
Auxin is commonly known to induce LR formation as suggested in different auxin
biosynthesis and signalling mutants in Arabidopsis and tomato [
105
,
106
]; however, there
are reports suggesting that exogenous auxin application (25nM) reduced the induction
of LRs [
107
]. In the roots, a regular pattern of root branching depends on the oscilla-
tory gene activity that determines prebranch sites. A recent report suggests that local
auxin source in the root cap is derived from the auxin precursor indole-3-butyric acid
(IBA), which regulates the oscillation amplitude and therefore determines the origin of the
prebranch site. An IBA-regulated gene, MEMBRANE-ASSOCIATED KINASE REGULA-
TOR4 (MAKR4), converts the prebranch site into a regular spacing of lateral organs [
108
].
LBD16/ASYMMETRIC LEAVES2-LIKE18 (ASL18), which is a direct downstream target
of SOLITARY ROOT (SLR)/IAA14-ARF7-ARF19 auxin signalling module, regulates LR
development possibly through transcriptional regulation through its related LBD/ASLs,
causing nuclear migration and asymmetric cell division for LR initiation and primordium
development (Figure 3) [
109
]. A very recent report shows the role of ABCB transporter
in LR emergence [
110
]. The report suggests that loss of ABCB21 reduces rootward auxin
transport and delays LR emergence [
110
]. These results support a primary role for ABCB21
in regulating auxin distribution supplementary to the primary ABCB auxin transporters
ABCB1 and 19 [
110
]. It is a well-established fact that ARF7 and the LATERAL ORGAN
BOUNDARIES DOMAIN (LBD) module are required for Arabidopsis LR development. A
recent report suggests that PR1 homolog PRH1 regulates LR development in Arabidopsis
via the auxin signalling pathway. It was shown that the expression of PRH1 was induced
upon auxin, and mutants of PRH1 had fewer LRs. Additionally, PRH1 was shown to be
a direct transcriptional target of ARF7 and LBDs [
111
]. PRH1 might then regulate the
expression of EXPANSIN (EXP) genes, which then affect cell wall loosening (Figure 3).
Int. J. Mol. Sci. 2021,22, 5508 11 of 29
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 30
3.2. Lateral Roots
Auxin is commonly known to induce LR formation as suggested in different auxin
biosynthesis and signalling mutants in Arabidopsis and tomato [105,106]; however, there
are reports suggesting that exogenous auxin application (25nM) reduced the induction of
LRs [107]. In the roots, a regular pattern of root branching depends on the oscillatory
gene activity that determines prebranch sites. A recent report suggests that local auxin
source in the root cap is derived from the auxin precursor indole-3-butyric acid (IBA),
which regulates the oscillation amplitude and therefore determines the origin of the
prebranch site. An IBA-regulated gene, MEMBRANE-ASSOCIATED KINASE REGU-
LATOR4 (MAKR4), converts the prebranch site into a regular spacing of lateral organs
[108]. LBD16/ASYMMETRIC LEAVES2-LIKE18 (ASL18), which is a direct downstream
target of SOLITARY ROOT (SLR)/IAA14-ARF7-ARF19 auxin signalling module, regu-
lates LR development possibly through transcriptional regulation through its related
LBD/ASLs, causing nuclear migration and asymmetric cell division for LR initiation and
primordium development (Figure 3) [109]. A very recent report shows the role of ABCB
transporter in LR emergence [110]. The report suggests that loss of ABCB21 reduces
rootward auxin transport and delays LR emergence [110]. These results support a pri-
mary role for ABCB21 in regulating auxin distribution supplementary to the primary
ABCB auxin transporters ABCB1 and 19 [110]. It is a well-established fact that ARF7 and
the LATERAL ORGAN BOUNDARIES DOMAIN (LBD) module are required for Ara-
bidopsis LR development. A recent report suggests that PR1 homolog PRH1 regulates LR
development in Arabidopsis via the auxin signalling pathway. It was shown that the
expression of PRH1 was induced upon auxin, and mutants of PRH1 had fewer LRs. Ad-
ditionally, PRH1 was shown to be a direct transcriptional target of ARF7 and LBDs [111].
PRH1 might then regulate the expression of EXPANSIN (EXP) genes, which then affect
cell wall loosening (Figure 3).
Figure 3. Regulation of LR development by various hormones and their crosstalk. Auxin via SLR4/ARF7-ARF9 signalling
module promotes LR development. Auxin-induced expression of PRH1 is dependent on ARF7 and LBD29. PRH1 may
promote LR development by regulating the expression of EXP genes that promote cell wall loosening. SLR4/ARF7-ARF9
signalling module also activates the expression of LBD16/ASL18 that are involved in the symmetry breaking of LR
founder cells for LR initiation and primordium development. JA positively regulates lateral root formation by modulat-
Auxin
SLR4/IAA14
ARF19ARF7
LBD29
PRH1
EXPs
LBD16/ASL18
Nuclear
migration
Asymmetric
Cell division
La tera l root deve lopment
CK
Polar auxin t ransport
JA
JA-Ile ERF109
YUC9
ASA1
Auxin formation and
redis tribution
BR
Figure 3.
Regulation of LR development by various hormones and their crosstalk. Auxin via SLR4/ARF7-ARF9 signalling
module promotes LR development. Auxin-induced expression of PRH1 is dependent on ARF7 and LBD29. PRH1 may
promote LR development by regulating the expression of EXP genes that promote cell wall loosening. SLR4/ARF7-ARF9
signalling module also activates the expression of LBD16/ASL18 that are involved in the symmetry breaking of LR founder
cells for LR initiation and primordium development. JA positively regulates lateral root formation by modulating auxin
biosynthesis and homeostasis. JA induces the expression of ASA1 both through COI1 and ERF109. ERF109 also induces
the expression of another auxin biosynthetic gene, YUC9. BR regulates lateral root development by interacting with auxin.
At low concentration, BR modulates auxin transport and promotes lateral root development. CK negatively regulates LR
initiation and development by inhibiting auxin carriers. Solid black arrows indicate confirmed pathway. Dotted arrow
indicates pathways for which there is little or no evidence.
Although the optimization of LR development is primarily governed by auxin, vari-
ous studies highlight the role of CKs in LR patterning and growth [
60
,
63
,
112
]. Previous
reports demonstrate that change in the CK levels, either through increased endogenous
signalling or through external application, inhibits LR production [
113
,
114
]. In line with
this, a decrease in CK signalling increased LRs [
58
,
65
,
71
,
115
]. CK insensitive mutants have
increased LR formation as a result of increased auxin perception [
114
], whereas LR forma-
tion in ahk triple mutant was severely impaired as a result of a decrease in cell division [
70
].
The inhibition of LR by CK is brought indirectly by changing auxin transport by disrupting
PIN1 localization (Figure 3) [
116
]. This results in a change of cell division pattern of LRFC
in the outer layer from undergoing periclinal division [
113
]. Upon application of exogenous
CK, a rapid modulation in PIN1 dynamics on the plasma membrane showed the role of
CK in controlling auxin flow. CK also led to the post-transcriptional degradation of PIN1
proteins through targeted lysis in vacuoles [
116
]. The endocytic trafficking of PIN proteins
is regulated by AHK4 and type-B ARR (ARR2 and ARR12) [
117
]. Plants perturbed in their
perception and/or signalling of CKs have large meristem sizes [
71
] and a higher number
of LRs [
65
], and exhibit increased root branching phenotype [
58
], implying the important
role of CKs in maintaining homeostasis in regulating RSA [
66
]. A recent report claims
that the impact of CK on LR elongation is tissue- and concentration-specific as low CK
levels reduced the length of the laterals in the apical/proximal region of the root system.
By contrast, high levels of CK reduced the length along the entire root system, but with
Int. J. Mol. Sci. 2021,22, 5508 12 of 29
a more pronounced effect in the apical zones [
63
]. Another interesting report introduces
TRANSPORTER OF IBA1 (TOB1), as a new player in LR development. Michniewicz and
co-workers show that the TOB1 gene is responsible for transport of IBA to the cytoplasm
and converts it into active IAA. They also show that it is a direct target of the CK response
pathway, and mutants altered in TOB1 show an increased number of emerged laterals.
Thus, TOB1 acts as a link between CK signalling and auxin to fine-tune RSA [118].
Apart from their role in inhibiting LR growth, CKs regulate the spacing of LRP
along the PR in Arabidopsis, as disruption of CK signalling genes led to irregularities in
spacing of consecutive LRP sites [
119
]. High local endogenous CK levels in pericycle cells
neighbouring the pre-existing LRs inhibit LRP initiation at proximity to existing ones. This
spacing is regulated via blockage of pericycle founder cells from G2 to M phase transition
by CK [60,119,120].
BRs are also one of the major factors regulating LR development. BRs modulate LR
development primarily via interacting with auxin. BR signalling mutant brassinosteroid
insensitive 1 (bri1) has been reported to have a decreased number of LRs, suggesting the
possible involvement of BRs in LR development. Furthermore, a low concentration of
BR has a positive effect on LR numbers. BRs promote the initiation of LR primordia by
increasing acropetal auxin transport (Figure 3) [
121
]. At low levels of auxin, BRs and auxin
exhibited a synergistic effect on LR formation, which was inhibited by NPA, an auxin trans-
port inhibitor [
121
]. However, higher concentrations of BRs have a negative effect on LR
formation [
122
]. Higher concentrations of BRs have been shown to induce the expression
of several AUX/IAAs, which play a vital role in LR formation [
123
]. These reports suggest
that high concentrations of BRs inhibit LR formation, possibly via inhibiting auxin sig-
nalling through these AUX/IAAs [
81
]. CK has been shown to antagonise the effect of BRs
on LR development. CK receptor double mutants ahk2ahk3 showed increased sensitivity
to BRs in LR elongation, indicating the antagonistic actions of these two hormones in LR
development [113].
Various reports strongly suggest the role of JA in LR development. It was found that
the promoters of all four members of ALLENE OXIDECYCLASE (AOC), a JA biosynthesis
gene, were highly expressed in LR primordia, suggesting the involvement of JA in LR
formation and development [
124
]. Several reports show the positive effect of JA on LR
initiation and growth by affecting auxin biosynthesis and homeostasis. It was found
that exogenous application of MeJA represses LRP initiation in asa1-1. Transcriptional
studies further validated that MeJA induces the expression of ASA1 in a COI1-dependent
manner that ultimately leads to increased local auxin accumulation in the root basal
meristem and, as a result, promotes LR formation (Figure 3) [
95
]. Another mechanism by
which JA promotes LR formation is by inducing the expression of ETHYLENE RESPONSE
FACTOR 109 (ERF 109), which then binds to the GCC boxes in the promoters of ASA1
and YUCCA 9 (YUC9) (Figure 3) [
125
]. COI1 is also involved in JA-induced pericycle
cell activation and LR formation, positioning, and emergence on bends and requires a
canonical auxin signalling pathway [
126
]. A very recent report highlights the negative role
of JA in regulating auxin induced LR formation, but independently of COIreceptor [
127
].
Higher concentrations of (
−
) JA and (+) JA were shown to counter the promontory effect of
auxin by stabilizing DII-VENUS and suppressing the expression of PUCHI and LATERAL
ORGAN BOUNDARIES-DOMAIN 29 (LBD29), involved in LR formation [127].
SA has been shown to enhance root biomass and a number of emerged LRs in Catha-
ranthus roseus [
128
]. The distance between the root tip and the first emerged LR primordia
was also decreased, which could be due to SA-induced reduction in the level of CK in the
root cap or lower CK sensitivity in apical cells of the root. Femtomolar (fM) concentrations
of SA displayed a larger root cap with a higher number of columella cells [
128
].SA also
enhanced the LR formation in a dose-dependent manner [
100
]. Armengot and co-workers
revealed that the mutants of CK2, a Ser/Thr kinase, accumulated high levels of SA, which
further resulted in the impediment of LR growth [
101
]. Pasternak et al. 2019 reported
inhibition of LR number with increasing concentrations of SA [
100
]. Similarly, the appli-
Int. J. Mol. Sci. 2021,22, 5508 13 of 29
cation of 20
µ
M SA or 40
µ
M SA to Arabidopsis seedlings inhibited LR organogenesis by
changing the auxin homeostasis through the cellular redistribution of PIN proteins [104].
3.3. Adventitious Roots
Lateral and adventitious roots mostly share common regulatory networks except for a
few regulatory mechanisms that distinguish lateral and AR pathways. AUXIN RESPONSE
FACTOR 7 (ARF7) and AUXIN RESPONSE FACTOR 19(ARF19) are key molecular play-
ers in the regulation of LR initiation and transcriptionally activate LATERAL ORGAN
BOUNDARIES-DOMAIN (LBD) genes. In the case of ARs, molecular functions of ARFs
are overtaken by WUSCHEL RELATED HOMEOBOX 11 (WOX11) transcription factor.
WOX11 responds to wound-induced auxin in the procambium and its surrounding cells
and works redundantly with its homologous protein WUSCHEL RELATED HOMEOBOX
12 (WOX12) to activate downstream LBD16 and 29, resulting in the fate transition from
procambium to root founder cell [
129
]. However, a few reports suggest that ARF7/19 also
govern the formation of adventitious roots from hypocotyls, which precludes the discrimi-
nation of molecular pathways of LR and adventitious roots [
130
]. Auxin transporters are
also important for AR development. For example, an ATP-binding cassette efflux family,
ABCB19, has been linked to AR formation [
131
]. Other members of the same transporter
are found to transport IBA [
132
,
133
], which has been tested to have the highest ability to
induce adventitious rooting [
134
]. Other hormones such as ethylene have been shown
to regulate the positioning of AR primordia [
135
]. In the fern, Ceratopteris richardii, an
intermediate clade WOX (IC-WOX) gene, CrWOXA, which is exclusively expressed in
root founder cells during LR and AR initiation, regulates AR organogenesis. CrWOXA
is a direct target of the auxin signalling pathway, which in turn may directly activate the
WUSCHEL-clade WOX (WC-WOX) gene, CrWUL [
136
]. A similar case is observed in
Arabidopsis, where WOX5/7 expression is directly regulated by WOX11/12 required for
root founder cell division during AR initiation [
137
]. AR initiation is mediated by ARF6,
8, and 17 modules, which control the expression of GH3.3,GH3.5, and GH3.6 (Figure 4).
ARF 6 and 8 are positive regulators, whereas ARF17 is a negative regulator, thus altering
auxin homeostasis. Thus, a complex regulatory network exists that fine-tunes AR initiation
(Figure 4) [138,139].
Research conducted on crop plants indicates the role of CK in concert with auxin in
the regulation of AR induction and development. In apple rootstocks, N6-benzyladenine
(BA) was shown to inhibit AR formation via restricting root primordium growth [
140
]. In
tomato, zeatin levels increased during AR induction and extension, which was reduced
after IAA treatment [
141
]. CK also negatively affect PIN, YUCCA6 and LAX3 expression,
leading to reduced auxin flow and hence, AR repression (Figure 4) [
135
]. Transcriptomic
studies dealing with poplar softwood cuttings revealed that PtARRs were significantly
downregulated during a time course of 6 h that was concomitant with an increase in
auxin signalling genes, depicting a general antagonism between auxin and CK during AR
induction [
142
]. The PtARR13 (a positive CK regulator) overexpressing shoots exhibited
differential expression of two most important genes; PLEIOTROPIC DRUG RESISTANCE
TRANSPORTER9 (PDR9) and TINY, which are induced via auxin and ethylene, respec-
tively, in normal WT excised shoots [
142
], implying that CK inhibits AR development
through interactions with other hormonal networks (Figure 4). In Arabidopsis, the CK
receptor ahk4 mutants, also known as wooden leg-3 (wol-3), and the higher order mutant
ahk2ahk3ahk4 exhibited enhanced AR formation on the hypocotyl [
70
,
143
]. Phenotypic
analysis of plants overexpressing the CK-degrading enzymes such as the CYTOKININ
OXIDASE/DEHYDROGENASE (AtCKX) gene family also demonstrates the role of CKs
in AR formation. The 35S:AtCKX2 and 35S:AtCKX4 lines displayed enhanced AR, which
was almost 7 times as much in comparison to the WT [
58
]. However, in Vigna callus,
kinetin alone was able to induce rhizogenic activity of the callus in terms of AR forma-
tion [
144
] pointing out the fact that distinct signals in different plant species determine
specific outcomes in terms of AR induction and formation. BR has been reported to induce
Int. J. Mol. Sci. 2021,22, 5508 14 of 29
adventitious rooting in cucumber at lower concentrations through a nitric oxide-dependent
pathway, whereas a higher concentration was inhibitory [145].
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 14 of 30
is directly regulated by WOX11/12 required for root founder cell division during AR ini-
tiation [137]. AR initiation is mediated by ARF6, 8, and 17 modules, which control the
expression of GH3.3, GH3.5, and GH3.6 (Figure 4). ARF 6 and 8 are positive regulators,
whereas ARF17 is a negative regulator, thus altering auxin homeostasis. Thus, a complex
regulatory network exists that fine-tunes AR initiation (Figure 4) [138,139].
Figure 4. Regulation of AR development by various hormones and their crosstalk. Auxin controls
AR initiation by activating ARF6 and ARF8, leading to downregulation of COI1-mediated JA sig-
nalling. GH3.3, GH3.5, and GH3.6 are regulated by ARF6, ARF8, and ARF17. The 3 GH3s control
JA homeostasis. JA level controls JA-Ile levels. JA-Ile negatively regulates AR development by ac-
tivating COI1-dependent signalling. Feedback regulation by DAO1 is activated by JA signalling,
which then regulates IAA homeostasis. SA at low concentration promotes AR development,
whereas at higher concentrations it inhibits AR development. CK regulates auxin homeostasis by
negatively affecting auxin carriers PIN, LAX, and YUCCA1 genes, thereby regulating adventitious
rooting through these pathways. In poplar cuttings, CK signalling activates PtARR13, which re-
presses AR formation by promoting the expression of PDR9 and inhibiting the expression of TI-
NY-Like TF. Solid black arrows indicate confirmed pathway. Dotted arrow indicates pathways for
which there is little or no evidence. Blue arrow indicates low level of SA and green arrow indicates
high level of SA.
Research conducted on crop plants indicates the role of CK in concert with auxin in
the regulation of AR induction and development. In apple rootstocks, N6-benzyladenine
(BA) was shown to inhibit AR formation via restricting root primordium growth [140]. In
tomato, zeatin levels increased during AR induction and extension, which was reduced
after IAA treatment [141]. CK also negatively affect PIN, YUCCA6 and LAX3 expression,
leading to reduced auxin flow and hence, AR repression (Figure 4) [135]. Transcriptomic
studies dealing with poplar softwood cuttings revealed that PtARRs were significantly
downregulated during a time course of 6 h that was concomitant with an increase in
auxin signalling genes, depicting a general antagonism between auxin and CK during AR
induction [142]. The PtARR13 (a positive CK regulator) overexpressing shoots exhibited
differential expression of two most important genes; PLEIOTROPIC DRUG RESISTANCE
TRANSPORTER9 (PDR9) and TINY, which are induced via auxin and ethylene, respec-
ARF6 ARF8
GH3.3, GH3.5, GH3.6
JA-AA JA-Ile COI1
Adventitious rooting
PtARR13
PDR9 TINY-like
DAO1
CK Ethylene
Auxin
JA
YUCCA
PIN1
LAX
ARF17
SA
Figure 4.
Regulation of AR development by various hormones and their crosstalk. Auxin controls AR initiation by activating
ARF6 and ARF8, leading to downregulation of COI1-mediated JA signalling. GH3.3, GH3.5, and GH3.6 are regulated by
ARF6, ARF8, and ARF17. The 3 GH3s control JA homeostasis. JA level controls JA-Ile levels. JA-Ile negatively regulates AR
development by activating COI1-dependent signalling. Feedback regulation by DAO1 is activated by JA signalling, which
then regulates IAA homeostasis. SA at low concentration promotes AR development, whereas at higher concentrations it
inhibits AR development. CK regulates auxin homeostasis by negatively affecting auxin carriers PIN,LAX, and YUCCA1
genes, thereby regulating adventitious rooting through these pathways. In poplar cuttings, CK signalling activates PtARR13,
which represses AR formation by promoting the expression of PDR9 and inhibiting the expression of TINY-Like TF. Solid
black arrows indicate confirmed pathway. Dotted arrow indicates pathways for which there is little or no evidence. Blue
arrow indicates low level of SA and green arrow indicates high level of SA.
JA differentially regulates AR formation in different species. In Arabidopsis, JA
negatively regulates adventitious rooting via COI1 and MYC2/3/4 dependent JA signalling
pathway by altering the JA-Ile homeostasis. jar1-1,coi1-16,myc2,myc3, and myc4 form
numerous adventitious roots as compared to the WT, and this response was dependent
on ARF 6 and ARF8 (Figure 4) [
139
]. Additionally, GH3.3, GH 3.5, and GH3.6 catalyse the
production of inactive forms of JA, such as JA-Asp, JA-Trp, and JA-Met, thus controlling JA
homeostasis and AR initiation (Figure 4) [
139
]. However, JA enhanced the AR formation in
leafy cuttings of Petunia hybrid [
146
]. Very recently, Druege and co-workers established that
early wound-induced JA accumulation leads to enhanced IAA accumulation and hence
stimulates AR production in Petunia cuttings [
147
]. The effect of JA in AR development
depends on experimental conditions. At low sub-micromolar concentrations, MeJA has
been shown to promote AR development in dark grown hypocotyls of Arabidopsis and
thin cell layers of N. tabacum when applied together with IBA and kinetin [
148
]. Recently
Lakehal and co-workers proposed that TIR1 and AUXIN SIGNALING F-BOX 2 (AFB2)
interact with IAA6, IAA9, and IAA17 to control JA homeostasis and AR initiation in
Arabidopsis [
138
]. They also showed that a feedback circuit exists between IAA and JA that
Int. J. Mol. Sci. 2021,22, 5508 15 of 29
is mediated by DIOXYGENASE FOR AUXIN OXIDATION (DAO1) and COI1-dependent
JA signalling. DAO1 catalyses the conversion of IAA to oxindole-3-acetic acid (oxIAA),
hence leading to reduced free IAA levels and AR initiation in Arabidopsis. They also
showed that the expression of DAO1 is induced by JA signalling (Figure 4) [149].
SA has also been shown to promote the formation of secondary adventitious roots
in Arabidopsis seedlings at a lower concentration (30
µ
M) and showed inhibition of AR
growth at concentrations higher than 30
µ
M SA [
100
]. At a concentration of 200
µ
M, SA ini-
tiated AR formation in mung bean (Phaseolus radiatus L.) hypocotyl cuttings, with maximum
effect at 400
µ
M SA. Further increases in SA concentration inhibited the response. Treat-
ment of mung bean hypocotyl cuttings with N, N’-dimethyl thiourea (DMTU), an H
2
O
2
scavenger, led to a decline in the number of ARs. Similarly, diphenyleneiodonium (DPI),
a specific inhibitor of membrane-linked NADPH oxidase, also restricted AR formation,
suggesting H2O2functions synergistically with SA to induce adventitious rooting [150].
3.4. Root Hair
Phytohormone auxin promotes the growth of RH [
151
]. ARF5/7/8/19 bind to the
promoter of RSL4 to control its expression in trichoblasts [152]. ARF19 was also shown to
regulate the expression of RSL2(Figure 5) [
153
]; however, overexpression of auxin repres-
sors ARFs in RH (ARF1-4,ARF9-11, and ARF16) suppressed RH growth (Figure 5) [
154
],
suggesting differential activity of ARFs. Among other phytohormones, it was found that
auxin and ethylene act synergistically in regulating RH growth. A transcriptome study sug-
gests that almost 90% of the auxin-regulated genes underlying RH growth were synergistic
to ethylene [
25
]. Moreover, other hormones such as JA, BR, and strigolactone pathways
may regulate root hair growth possibly through interconnections with the auxin/ethylene
signalling pathways [
155
]. BR inhibits root hair growth. The expression of root hair-related
AUX/IAA (such as AXR2, AXR3, and SLR) was increased by the application of EpiBL and
repressed in the BR-insensitive bri1 mutant, suggesting a possibility that BR may suppress
root hair growth through the suppression of auxin signalling. JA positively regulates
root hair growth. The interconnection of JA and auxin was shown as axr2 mutants were
found to be resistant to exogenous JA in primary root inhibition, and JA was also shown to
promote auxin biosynthesis. However, no direct evidence supports idea that JA regulates
root hairs through auxin (extensively reviewed by Lee and Cho, 2013). In RH cells, auxin
promotes membrane depolarization in a concentration- and pH-dependent manner that is
strongly dependent on AUX1-mediated auxin transport. AUX1-mediated auxin transport
involved SCF TIR1/AFB-type Ca2+ signalling in the RH as treatment with SCFTIR1/AFB
inhibitor auxinole blocked AUX1 transport and IAA-triggered calcium signals [
156
]. Using
3D modelling, Jones et al. 2009 showed that AUX1-dependent auxin transport via non-hair
cells maintains the auxin flow in the development of RH [
157
]. In a recent study, auxin was
found to govern the transcriptional regulation of a cell wall receptor protein ERULUS (ERU)
in an ARF7- and 19- dependent manner (Figure 5). ERU is a member of the Catharanthus
roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) subfamily of cell wall receptors and
regulates RH cell wall composition and modulates pectin dynamics [
26
]. MEDIATOR18
(MED18) protein, through the modulation of auxin signalling and transport, influences the
growth of PR, LR, and RH development [
158
]. The energy status of plants also affects RH
development. In a recent study, glucose-activated TOR was found to phosphorylate and
stabilize PIN2 at the plasma membrane, which results in the shootward auxin transport to
the RH [159,160].
Int. J. Mol. Sci. 2021,22, 5508 16 of 29
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 17 of 30
regulating the distribution of hair (H) and non-hair (N) cells in the root epidermis. Root
hair formation is determined by transcription factor complex formation and lateral
communication between H- and N-cells. At high BR concentrations, BIN2 regulates this
distribution primarily via the phosphorylation of two major transcription factors, EN-
HANCER OF GLABRA3 (EGL3) and TRANSPARENT TESTA GLABRA 1 (TTG1), lead-
ing to functional TF complex and subsequent GL2expression and determination of the
non-hair cell fate in all epidermal cells (Figure 5) [168]. It has also been reported that ex-
pression of BRI1 under protophloem-specific promoters rescued root meristem pheno-
typic defects of bri1 brl1 brl3 triple receptor mutants [169]. All of these results suggest that
not only optimal concentration of BR but tissue-specific expression of BR signalling
components also play a crucial role in root development.
Figure 5. Regulation of RH development by various hormones and their crosstalk.BR positively regulates the expression of WER,
GL2 (negative regulator of RH development) and inhibits BIN2, which is then unable to phosphorylate GL3/EGL3 and TTG1.
Presence of BR promotes functional TF complex formation between TTG1, GL3/EGL3, and WER, promoting GL2 expression, leading
to inhibition of the RH development pathway. CK induces TF ZFP5, which promotes the expression of CPC (positive regulator of
RH development), leading to the formation of functional TF complex of TTG1, GL3/EGL3, and CPC. The complex inhibits the ex-
pression of GL2, thus causing activation of the RH development pathway. The presence of auxin liberates the ARFs from
AUX/IAAs. The ARFs show different specificity to RH development. ARF4-11 inhibits, whereas ARF6 promotes, the RH develop-
ment pathway. ARF5 directly interacts with the promotor of RSL4, leading to RH development. Auxin also directly regulates the
expression of RSL4 target gene ERU via ARF7/ARF19. JA positively influences RH growth via ET signalling. Solid black arrows
indicate confirmed pathways.
BR
BIN2
TTG1 WER
GL3/EGL3
GL2
RHD6/RSL1
RSL2
RSL4
ERU
Root hair development
Auxin
ARF5 ARF7
ARF19
JA
Ethylene
signalling
CK
ZFP5
CPC TTG1 GL3/EGL3
ARF4-11
Figure 5.
Regulation of RH development by various hormones and their crosstalk.BR positively
regulates the expression of WER, GL2 (negative regulator of RH development) and inhibits BIN2,
which is then unable to phosphorylate GL3/EGL3 and TTG1. Presence of BR promotes functional
TF complex formation between TTG1, GL3/EGL3, and WER, promoting GL2 expression, leading to
inhibition of the RH development pathway. CK induces TF ZFP5, which promotes the expression of
CPC (positive regulator of RH development), leading to the formation of functional TF complex of
TTG1, GL3/EGL3, and CPC. The complex inhibits the expression of GL2, thus causing activation of
the RH development pathway. The presence of auxin liberates the ARFs from AUX/IAAs. The ARFs
show different specificity to RH development. ARF4-11 inhibits, whereas ARF6 promotes, the RH de-
velopment pathway. ARF5 directly interacts with the promotor of RSL4, leading to RH development.
Auxin also directly regulates the expression of RSL4 target gene ERU via ARF7/ARF19. JA positively
influences RH growth via ET signalling. Solid black arrows indicate confirmed pathways.
There are very few reports citing the development of RH by CK. CK promotes RH
formation at the differentiation zone regulating a similar set of RH-specific genes, as
observed for auxin and ethylene (Zhang et al. 2016). CK independently regulate RH
formation to that of auxin and ethylene (Zhang et al. 2016). CK levels are also modulated
in RH of root nodule-forming species, such as in Medicago trancatula via several cytokinin
biosynthesis and signalling genes, including CYTOKININ RESPONSE 1(CRE1) and type A
ARRs such as MtARRA2,MtARRA8,MtARRA9, and MtARRA10 in response to nodulation
factors, indicating that CK signalling negatively impacts root epidermal infections [
161
,
162
].
In Arabidopsis, 6-Benzylaminopurine (BAP) treated seedlings showed enhanced RH
growth in response to low hormonal concentrations (10
µ
M) [
163
]. Apparently, this pro-
Int. J. Mol. Sci. 2021,22, 5508 17 of 29
found effect was auxin- and ethylene-independent, as similar results were achieved in axr1,
ethylene response 1 (etr1), and ethylene inhibitor Aminoethoxyvinylglycine (AVG) treated
seedlings, respectively (Zhang et al. 2016), while on the other hand, CKX2 overexpression
line (35S:CKX2) displayed abnormal short-hair phenotypes [
161
]. In addition, several
proteins and TFs regulate RH development, and are themselves orchestrated by upstream
phytohormonal networks. This is exemplified via molecular characterization of the C
2
H
2
zinc finger protein, ZINC FINGER PROTEIN 5 (ZFP5), whose mutant has fewer RHs than
the WT [
164
]; its expression is transcriptionally increased by BAP application (
Figure 5
).
In effect, ZFP5 itself induces the expression of another RH-localised RB MYB protein,
CAPRICE (CPC), involved in RH patterning by binding to its promoter, which then inhibits
negative regulators of RH development such as GLABRA2 (GL2) (Figure 5) [165].
In the case of RH, the relative spatial distribution of BRI1 plays a crucial role. Earlier,
it was reported that BRs play an essential role in the establishment of epidermal cell
fate in Arabidopsis roots. BRs convey the positional information of hair and non-hair
cells along the roots via BRI1 [
166
]. Later, it was found that expression of BRI1 in hair
cells has a promontory effect, whereas in the case of non-hair cells, its expression is
inhibitory [
167
]. Additionally, BRASSINOSTEROID-INSENSITIVE 2 (BIN2) also plays a
key role in regulating the distribution of hair (H) and non-hair (N) cells in the root epidermis.
Root hair formation is determined by transcription factor complex formation and lateral
communication between H- and N-cells. At high BR concentrations, BIN2 regulates
this distribution primarily via the phosphorylation of two major transcription factors,
ENHANCER OF GLABRA3 (EGL3) and TRANSPARENT TESTA GLABRA 1 (TTG1),
leading to functional TF complex and subsequent GL2 expression and determination of
the non-hair cell fate in all epidermal cells (Figure 5) [
168
]. It has also been reported
that expression of BRI1 under protophloem-specific promoters rescued root meristem
phenotypic defects of bri1 brl1 brl3 triple receptor mutants [
169
]. All of these results suggest
that not only optimal concentration of BR but tissue-specific expression of BR signalling
components also play a crucial role in root development.
JA and MeJA also positively influence RH growth via ethylene signalling (
Figure 5
) [
170
].
It was found that the effects of JAs were abolished in the ethylene-insensitive mutants
etr1-1 and etr1-3, or by ethylene biosynthesis inhibitors (AVG) [170].
3.5. Gravitropism
Auxin is the major hormone that drives the growth of an organ (PRs or LRs) towards
the gravity vector. The angle at which an organ grows with respect to the gravity vec-
tor is called the gravitropic set point angle (GSA) [
171
]. It is a well-established fact that
the distribution of auxin drives the root growth in response to gravity via the starch sta-
tolith hypothesis [
172
]. In response to gravity-stimulation of PRs, polarized localization
of PIN3 and PIN7 redirects auxin flux towards the lower side of the PR, thus, causing
differential cell elongation and root tip bending [
173
–
175
]. The dual role of auxin in grav-
ity perception and gravity response was demonstrated by Zhang and co-workers. They
reported thatTIR1-dependent auxin signalling module TIR1-AFB-IAA17 mediates starch
granule formation and gravitropic perception in root tips by regulating the expression of
starch granule synthesis genes PHOSPHOGLUCOMUTASE (PGM), ADENOSINE DIPHOS-
PHATE GLUCOSE PYROPHOSPHORYLASE (ADG1), and STARCH SYNTHASE 4 (SS4)
(
Figure 6A
) [
31
]. Downstream signalling of auxin reorients PR growth. Auxin influx and
efflux transporters distribute differential auxin gradient in the upper and lower sides of
the gravity-stimulated roots. The asymmetric distribution of auxin towards the lower
side of the root is modulated by AUX1 and PINs [
175
,
176
]. While auxin is the central
component of root gravitropic responses, ET is implicated in this process through crosstalk
with auxin, which also involves para-aminobenzoic acid (PABA) (Figure 6A) [
176
]. The
interference of ET in root graviresponses is mediated through its interaction with the auxin
pathway at the level of auxin biosynthesis and transport. In gravistimulated roots, PABA
promotes an asymmetric auxin response, which causes the asymmetric growth responsible
Int. J. Mol. Sci. 2021,22, 5508 18 of 29
for root curvature. This activity requires the auxin response transcription factors AUXIN
RESPONSE FACTOR7 (ARF7) and ARF19 as well as ethylene biosynthesis and signalling,
indicating that PABA activity requires both auxin and ethylene pathways (Figure 6A) [
176
].
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 18 of 30
JA and MeJA also positively influence RH growth via ethylene signalling (Figure 5)
[170]. It was found that the effects of JAs were abolished in the ethylene-insensitive mu-
tantsetr1-1andetr1-3, or by ethylene biosynthesis inhibitors (AVG) [170].
3.5. Gravitropism
Auxin is the major hormone that drives the growth of an organ (PRs or LRs) towards
the gravity vector. The angle at which an organ grows with respect to the gravity vector
is called the gravitropic set point angle (GSA) [171]. It is a well-established fact that the
distribution of auxin drives the root growth in response to gravity via the starch statolith
hypothesis [172]. In response to gravity-stimulation of PRs, polarized localization of PIN3
and PIN7 redirects auxin flux towards the lower side of the PR, thus, causing differential
cell elongation and root tip bending [173–175]. The dual role of auxin in gravity percep-
tion and gravity response was demonstrated by Zhang and co-workers. They reported
thatTIR1-dependent auxin signalling module TIR1-AFB-IAA17 mediates starch granule
formation and gravitropic perception in root tips by regulating the expression of starch
granule synthesis genes PHOSPHOGLUCOMUTASE (PGM), ADENOSINE DIPHOS-
PHATE GLUCOSE PYROPHOSPHORYLASE (ADG1), and STARCH SYNTHASE 4 (SS4)
(Figure 6A) [31]. Downstream signalling of auxin reorients PR growth. Auxin influx and
efflux transporters distribute differential auxin gradient in the upper and lower sides of
the gravity-stimulated roots. The asymmetric distribution of auxin towards the lower
side of the root is modulated by AUX1 and PINs [175,176]. While auxin is the central
component of root gravitropic responses, ET is implicated in this process through cross-
talk with auxin, which also involves para-aminobenzoic acid (PABA) (Figure 6A) [176].
The interference of ET in root graviresponses is mediated through its interaction with the
auxin pathway at the level of auxin biosynthesis and transport. In gravistimulated roots,
PABA promotes an asymmetric auxin response, which causes the asymmetric growth
responsible for root curvature. This activity requires the auxin response transcription
factors AUXIN RESPONSE FACTOR7 (ARF7) and ARF19 as well as ethylene biosynthe-
sis and signalling, indicating that PABA activity requires both auxin and ethylene path-
ways (Figure 6A) [176].
Figure 6. Regulation of PR and LR gravitropism by various hormones and their crosstalk. (A) PABA works upstream of
ET signalling, through which it regulates auxin biosynthesis and transport. This ultimately leads to ARF7/19 mediated
JA JA-Ile COI1 JAZ9
MYC2
CYP79B29
MYC2
MYC2
LAZY4
LAZY2
Auxin
homeost asis
LR
gravitropism
CK
PABA Et hylene Ethyle ne
signa lling
Auxin
biosynthes is
Auxin
transport
Auxin
gradient
BR
PIN2
Glc
TIR 1-AFB-IAA 17
SS4, ADG1, PGM
Star ch granule for mation
Root gr avitropic pe rception
EIN2 Asymmetr ic
root growth
ARF7
ARF19 Root gr avitropic
response
SA
Auxin dist ribution
JA
JA-Ile
ASA1
Auxin dis tribut ion
A
B
Auxin
Auxin
transport
Auxin
sig nalling
FLP
PIN3/7
MYB88
Figure 6.
Regulation of PR and LR gravitropism by various hormones and their crosstalk. (
A
) PABA works upstream of ET
signalling, through which it regulates auxin biosynthesis and transport. This ultimately leads to ARF7/19 mediated auxin
activity effects on asymmetric growth promotion followed by root gravitropic response. Glc acts via BR to regulate root
gravitropism. BR regulates root gravitropism by interacting with auxin via PIN2 distribution. Auxin signalling module
TIR1-AFB-IAA17 mediates starch granule formation and gravitropic perception by inhibiting the expression of PGM,ADG1,
and SS4. JA influences gravitropic response by modulating auxin levels through SA1 induction. SA affects auxin transport
and redistribution, leading to gravitropic bending. (
B
) The presence of JA activates COI1-mediated JA signalling that causes
proteasomal degradation of JAZ9, leading to the liberation of MYC2, which then binds to the promotors of CYP79B2 and
LAZ2 and LAZY4, leading to changes in auxin homeostasis and LR gravitropism. Auxin transport and signalling modules
are essential to regulate vertical orientation of LRs. The binding of FLP and MYB88 TFs leads to transcriptional activation
of PIN3 and PIN7, which then lead to gravitropic bending of LRs. CK perturbs auxin homeostasis, leading to horizontal
branching of LRs. Solid black arrows indicate confirmed pathways.
Biochemical and computational analyses have revealed that besides auxin, CK also
influences gravity responses in plants [
112
,
177
,
178
]. The combinatorial effect of auxin-CK
on root gravitropic responses is observed at the protein transport and differential expression
levels of PINs [
178
]. Several mutant lines defective in endogenous CK perception and
signalling such as ahk3 and higher-order mutants; ahk2 ahk3 ahk4,ahp1 ahp2 ahp3 ahp4 ahp5,
arr1 arr10 arr12, and the CK degrading lines 35S:AtCKX2 and 35S:AtCKX3, had higher
root gravitropic angles than the WT [
178
]. The expression of PIN3 was also shown to be
significantly lower in these lines as compared to the WT in the root tip plasma membrane
of columella cells. However lateral expansion of PIN7 levels was also observed in the
columella cells, although the intensity was comparable to the WT [
178
]. It is known
that CK alters auxin sensitivity and transport in root columella cells. AUX1 signals were
diminished in 35S:AtCKX2 and 35S:AtCKX3 lines, suggesting endogenous CKs regulate
AUX1 in root tips [
178
]. Root caps cells were also shown for their potential in producing
CKs upon gravistimulation in Arabidopsis, indicating the importance of de novo CK
synthesis [
179
,
180
]. The ARR5 exhibited an asymmetrical pattern and redistribution at the
lower side following a 90 degree shift in root orientation [
180
]. The tips aligned towards
Int. J. Mol. Sci. 2021,22, 5508 19 of 29
BAP-applied sites, causing a discernible bending and triggering an inhibition in root
elongation during gravity sensing [180].
There are several reports indicating the involvement of BR signalling in the regulation
of root gravitropic responses. Exogenous BR application is known to increase gravitropic
curvature of PRs [
181
,
182
]. BR signalling mutants showed reduced gravitropic curvature,
whereas transgenic plants overexpressing BRI1 were hypersensitive to gravity. BR and
auxin show a synergistic effect on gravitropic curvature at low concentrations of auxin
primarily via increasing acropetal and basipetal auxin transport [
181
,
182
]. BR has also
been found to promote the activity of RHO-RELATED PROTEIN FROM PLANTS 2 (ROP2)
GTPase, resulting in enhanced polar accumulation of PIN2 protein and thereby increased
gravitropic response [
182
]. However, high concentrations of auxin antagonise BR-induced
root gravitropic curvature and vice versa [
183
]. Exogenous BR could also affect gravitropic
responses by regulating the actin cytoskeleton and PIN2 localization pattern, and thereby
the auxin gradient in a way similar to auxin [
184
]. A recent report indicated that BR
antagonises PIN2 endocytosis [
185
]. BR-mediated modulation of endocytic sorting and
distribution of PIN2 plays a crucial role in the formation of lateral PIN2 gradient in
gravistimulated roots. This lateral gradient regulates auxin signalling in gravistimulated
roots and thereby root growth deviation (Figure 6A) [
185
]. In addition to this, BR plays a
key role in glucose (Glc) mediated root directional responses (Figure 6A) [
186
]. Exogenous
BR dramatically increased Glc-mediated directional responses. Additionally, BR signalling
mutant bri1-6 showed reduced root deviation in the presence of Glc, whereas bzr1-1D
showed increased root deviation in thepresence of Glc. Glc enhances BR signalling by
increasing the endocytosis of BRI1. It was found that BR modulates Glc-induced root
deviation responses by regulating PAT (Figure 6A) [
186
]. A recent report has implicated
the involvement of BR signalling in root gravitropic response in maize. Treatment with BR
resulted in more curvature of PRs of maize [187].
JA is also found to be a crucial regulator of root gravitropism. Studies on rice coleop-
tiles revealed an increased amount of JA upon gravistimulation. In addition, a JA gradient
was formed opposite to the internal auxin gradient across the stimulated organ during a
gravitropic response that worked in an IAA manner. Consistent with these observations,
a JA-deficient rice mutant, hebiba, bent slowly upon gravitropic stimulus, thus, suggest-
ing that JA might accelerate the bending response [
188
]. Moreover, Trp conjugates of
JA (JA-Trp) that act as IAA antagonists have been reported to cause agravitropism in a
dose-dependent fashion. The response is TIR1-dependent but COI1-independent [
189
].
JA also influences gravitropism via ASA1, which further leads to changes in auxin home-
ostasis (Figure 6A). Thus, JA might work via auxin to regulate tropic responses in roots.
Another report demonstrates that MeJA impairs lateral auxin distribution in WT roots
upon gravistimulation and therefore slows root gravitropic response [190].
Administration of SA led to inhibition of gravitropic response in maize roots, which
was an indirect result of inhibition of ethylene biosynthesis by SA. In the recent studies
by Tan and co-workers, the effect of exogenous SA on various root growth parameters
was examined. They concluded from their studies that exposure of 20
µ
M or 40
µ
M SA
to Arabidopsis seedlings led to partial agravitropic roots in an NPR1-independent man-
ner [
104
]. SA also affected the auxin redistribution in the PRs and was involved in the
gravitropic bending via auxin efflux carrier PIN2. SA leads to the agravitropic response
via blocking PP2A activity, causing the cellular SA levels to increase, thereby resulting in
phosphorylation of PIN proteins and affecting auxin transport and redistribution in the
cell (Figure 6A) [
104
]. SA also modulates root gravitropism by affecting clathrin-mediated
endocytosis, as exposure to 50
µ
M SA in wild type Arabidopsis seedlings led to a reduction
in gravitropic root curvature. However, the clathrin heavy chain (chc2-2) exhibited less sensi-
tivity to SA-induced gravitropic response [
191
]. A recent study by Ke et al. 2021 reported
that high concentrations of SA impaired the root gravitropism in Arabidopsis seedlings via
condensation of PIN2 into hyperclusters through REMORIN 1.2 (REM1.2) dependent nan-
odomain compartmentalization. As a result, the movement of PIN2 by clathrin-mediated
Int. J. Mol. Sci. 2021,22, 5508 20 of 29
endocytosis and lateral diffusion was restricted, thereby interfering with the asymmetric
distribution of auxin, leading to the inhibition of gravitropic responses [192].
Not only the main root, but other parts of the RSA such as LRs also show grav-
itropism [
193
,
194
]. In such cases, the LRs partially suppress positive ortho-gravitropic
growth and grow radially to cover more area in the soil. Supplying exogenous auxin and
an endogenous increase in auxin levels causes LRs to grow at a more vertical GSA. Reports
by Roychoudhry and co-workers have shown that the entire TIR1/AFB-Aux/IAA-ARF-
dependent auxin signalling module is necessary to establish GSA in LRs (Figure 6B) [
194
].
Recently emerged LRs (stage I) show strong but transient PIN3 expression, presumably
limiting the strength of auxin redistribution in (stage II) LRs that are establishing their
GSA [
193
,
195
]. At later stages, the expression of PIN3 diminishes, and PIN4 and PIN7
are expressed to maintain vertical GSA [
193
]. While auxin promotes gravitropic bending
of LRs, CK function as an anti-gravitropic component that is shown to suppress cellular
elongation in the upper portion of stage II LRs, thus promoting the radial expansion of
RSA (Figure 6B) [
112
]. Apart from this, there are other molecular components, such as
LAZY gene family in Arabidopsis, that has also been reported to have a role in main-
taining GSA by controlling the distribution of PINs. LAZY (LZY)/DEEPER ROOTING
(DRO)/NEGATIVE GRAVITROPIC RESPONSE OF ROOTS (NGR) genes affect auxin flow
towards the lower side of the LR in gravistimulated LRs. The lzy1lzy2lzy3 mutant showed
an inverse auxin distribution and asymmetry in the PIN3 expression in the columella cells
of the LR, resulting in a horizontally placed LR [
196
,
197
]. A recent report by Sharma et al.
2020 also showed a promising involvement of JA-auxin machinery in the regulation of
LR angle via LAZY genes. JA signalling transcription factor MYC2 directly activates the
transcription of CYP79B2, an auxin biosynthetic gene, and LAZY2/4 in the regulation of
LR angle. In addition, JA treatment also affects auxin transport machinery and PIN2
localization in the stage II LR, leading to a downward LR orientation (Figure 6B) [
198
]. The
Arabidopsis R2R3-MYB transcription factor FOUR LIPS (FLP) and its paralog, MYB88, act
redundantly but differently in the regulation of primary and LR gravity responses. FLP
alone is responsible for the transcription regulation of PIN3 and PIN7 in gravity-stimulating
cells in the PRs, whereas FLP-MYB88 both act redundantly to control LR gravitropic set
point angle (GSA) (Figure 6B) [
5
]. The Arabidopsis FERONIA (FER) receptor kinase, which
integrates multiple hormonal and environmental signals, governs auxin-mediated LR and
root gravitropic responses. The fer-4 mutants showed increased LR branching and delayed
gravitropism, which was found to be associated with aberrant PIN2 polarity and an altered
polar auxin transport [
199
]. Previous reports revealed that NGR is necessary for the posi-
tive gravitropism of roots. A recent study suggests that NGR, a plasma membrane protein
exclusively expressed in the columella and LR cap cells, governs positive gravitropism
through regulation of auxin efflux carrier PIN3 localization and modulation of lateral auxin
flow in response to gravity [
200
]. A Lotus japonica mutant defective in LAZY3, a functional
ortholog of LAZY1, showed negative gravitropism in primary and LRs. This negative
gravitropic response in lazy3 mutant was due to reduced polar auxin transport [
201
]. In
Arabidopsis, DEEPER ROOTING 1 (DRO1) mutation causes horizontally placed LRs and
altered GSA, while DRO1 overexpression showed steeper LR angles [
202
]. In a recent re-
port, Atdro1 primary and LRs showed defects in establishing an auxin gradient in response
to gravity [
203
]. Collectively, all of these results indicate a promising involvement of active
auxin signalling and transport machinery in the regulation of root gravitropic responses
in LRs.
4. Conclusions and Future Prospects
The RSA is continuously modified by abiotic and biotic signals from the soil that
interact with various endogenous cues and turn them into different cellular responses.
Phytohormones affect several overlapping processes and their actions depend on specific
hormone combinations rather than on the action of a single hormone individually. This
review has focussed on various examples of crosstalk between auxin-JA, auxin-CK, etc.,
Int. J. Mol. Sci. 2021,22, 5508 21 of 29
suggesting that different hormones affect a number of common responses in plants [
95
,
96
].
Moreover, these interactions do not occur via a simple linear mechanism; rather, there
are many feedback loops and the involvement of various downstream activators and
repressors that drive a single development output robustly. For example, binding of
MYC2 transcription factor on the promoters of LAZY2,LAZY4, and auxin-responsive gene
CYP79B2 leads to gravitropic bending of LRs [
198
]. A recent review by Semeradova et al.
2020 summarizes that auxin transport machinery is post-translationally modified by multi-
ple hormones that enable the rapid modulation of the auxin flow to control plant growth
and development [
204
]. Additionally, several other reports as mentioned in this review
have further clarified how almost all plant hormones influence the rate of auxin homeosta-
sis [
95
,
118
,
149
], thus making it clear that auxin is indispensable in controlling all aspects of
the plant life cycle.
It is very essential to map key transcriptional and post translational pathways and
their genes that define the distinct nature of PR and LRs, as that might be the key in
understanding hormonal and developmental outputs. Another interesting area is to
pinpoint DNA polymorphisms that control a particular root trait of interest. This provides
a powerful approach to identify new root branching regulatory genes. A challenging step
will be to study the regulation of different root traits under more realistic conditions than
under highly aseptic growth conditions such as agar plates where we might miss novel
adaptive strategies employed by the plants to survive in their microenvironment. Thus,
there is a need to integrate systems biology-aided computational methods (SimRoot) [
205
]
and computer technology such as magnetic resonance imaging and microscale computed
tomography [
206
] that will not only enable us to integrate information related to the overall
organization of how roots sense various endogenous and environmental signals, and how
they turn them into cellular responses, but also assist in devising predictive models that
may identify the crucial regulators, hubs, and integrators involved in regulating RSA.
Such multiscale mechanistic insights will underpin efforts to develop crops with improved
root systems.
Author Contributions:
A.L. and M.S. (Manvi Sharma) conceptualized the article. M.S. (Manvi Sharma),
D.S., M.S. (Mohan Sharma), A.T., H.B.S., P.A., H.K.B. and B.N.S. wrote the original draft. M.S., H.B.S.,
and H.K.B. prepared the figures. A.L. and M.S. (Manvi Sharma) reviewed and finalized the article.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments:
The research in AL laboratory is supported by a project grant from Department
of Biotechnology, Government of India (Grant No. BT/HRD/NWBA/37/01/2015) and Core Grant
from the National Institute of Plant Genome Research. M.S., M.S., and D.S. duly acknowledge
research fellowships from National Institute of Plant Genome Research, Govt of India. P.A., H.B.S.,
and H.K.B. duly acknowledge research fellowships from Department of Biotechnology. A.T. duly
acknowledges research fellowship from University Grants Commission, Govt. Of India. The authors
acknowledge DBT-eLibrary Consortium (DeLCON) for providing access to e-resources.
Conflicts of Interest:
The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict of interest.
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