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REVIEW ARTICLE
Keywords
1-Aminocyclopropane-1-carboxylic acid.
1-methylcyclopropene; glutamate-like
receptor; hydrogen cyanide; L-a-(2aminoethoxyvinyl)glycine; pyrodoxal-5phosphate; root growth; root morphogenetic
programme; silver nitrate; a-aminoisobutyric
acid.
Correspondence
E. Le Deunff, INRA, UMR 950, Laboratoire
dEcophysiologie V
eg
etale, Agronomie &
Nutritions N, C & S, Universit
e de Caen BasseNormandie, F-14000 Caen, France.
E-mail: erwan.ledeunff@unicaen.fr
Editor
A. Weber
ABSTRACT
In the last decade, genetic and pharmacological approaches have been used to explore
ethylene biosynthesis and perception in order to study the role of ethylene and ethylene/auxin interaction in root architecture development. However, recent findings
with pharmacological approaches highlight the non-specificity of commonly used
inhibitors. This suggests that caution is required for interpreting these studies and
that the use of pharmacological agents is a double-edged tool. On one hand, non-specific effects make interpretation difficult unless other experiments, such as with different mutants or with multiple diversely acting chemicals, are conducted. On the other
hand, the non-specificity of inhibitors opens up the possibility of uncovering some
ligands or modulators of new receptors such as plant glutamate-like receptors and
importance of some metabolic hubs in carbon and nitrogen metabolism such as the
pyridoxal phosphate biosynthesis involved in the regulation of the root morphogenetic
programme. Identification of such targets is a critical issue to improve the efficiency of
absorption of macronutrients in relation to root the morphogenetic programme.
INTRODUCTION
Plants need to optimise nutrient ions and water acquisition to
grow and survive in their fluctuating environment. Root
exploratory and root hair systems show both a structural and a
functional plasticity in response to various environmental and
developmental cues. At the structural level, the root morphogenetic programme depends on the activity of the meristems of
primary and lateral roots and proceeds by the succession of cell
divisions, followed by cell expansion and differentiation. These
processes are under the control of various endogenous hormones such as ethylene and auxin for which the signalling
pathways can overlap (Dugardeyn & Van Der Straeten 2008;
Muday et al. 2012). However, the understanding of coordination between ethylene and auxin biosynthesis with the carbon
and nitrogen metabolism during the root morphogenetic programming remains elusive.
For instance, at cellular level, it is commonly admitted that
rapid cell elongation (minutes to hours) is induced by an auxin
signalling cascade that provokes cell wall loosening via action
on H+-ATPase and an inward rectifying K+ channel at the
plasma membrane (Hager 2003; Fraas et al. 2014). However,
because pharmacological treatments with ethylene or the ethy-
Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
Thus, it has been recently demonstrated by genetic and pharmacological approaches in Arabidopsis that ethylene negatively
modulates the activity of H+-ATPase by increasing the auxin
influx and/or auxin biosynthesis (Staal et al. 2011). These
results are in agreement with ethylene-induced activation of
several genes of auxin biosynthesis in roots (Stepanova et al.
2005, 2007, 2008). It has also been shown that ethylene is able
to promote acropetal auxin transport from the shoot to the
root tip and basipetal auxin transport towards the elongation
zone by stimulating the auxin influx carrier AUX1 and the
efflux carriers PIN2 (R
uzicka et al. 2007; Stepanova et al. 2007;
Swarup et al. 2007).
Although pharmacological treatments are still extensively
used in studies of ethylene effects on root growth, recent results
highlighted the non-specificity of commonly used inhibitors of
ethylene biosynthesis such as AVG and AOA (Leblanc et al.
2008; Soeno et al. 2010) and perception such as Ag2+ (Strader
et al. 2009). These results suggest that caution is needed in
using these inhibitors and that those pharmacological agents
are double-edged tools. On one hand, these non-specific
effects require an exclusive use of chemicals with a well-known
target such as ACC or to develop new approaches such as
chemical genomics to highlight new and more specific molecules. Thus, recently, a chemical genomic study allowed the
discovery of more specific inhibitors of the ACS enzyme with
quinazolinone backbone (called 9393, 9370 and 7303 compounds). These molecules were discovered by screening a collection of 10,000 small molecules able to suppress the
constitutive triple response of the ethylene overproducer
mutant eto1-4 (Lin et al. 2010). On the other hand, the
non-specificity of previous ethylene inhibitors opens up the
possibility to explore and uncover new signalling pathways or
metabolic hubs in primary metabolism involved the regulation
of the root morphogenetic programme. Indeed, although carbon of the cell wall structure and cellular components represents about 3040% of the root dry weight (mg carbonmg1
root DW) in seedlings, we still do not know how ethylene and
urea
Ornithine
CO2
Spermidine
Putrescine
Arginine
Spermine
H2O
CO2 + NH3
Agmatine
MTA
dSAM
MTA
CO2
Sam dcarboxylase
= SamDc
Proteins
synthesis
CO2
AVG, AOA
ACC synthase=ACS
S-AdoMe
ATP
PPi + Pi
Methionine
2-oxo-acid
ACC
+ PLP
Amino acid
Adenine
ATPMTR
Pi +
HCOO
cysteine
H2S
MTR-1-P
O2
ADP
GSH
-cyanoalanine
H 2O
Glutamate
Malonyl CoA
GACC
MACC
-glutamylTranspeptidase
=-GT
N-malonyl
transferase
Jasmonic
acid
JA-amino synthetase
=JAR1
JA-ACC
-cyanoalanine
Synthase
= CAS
H2O
Methionine
(Yang) cycle
KMB
O2 + ascorbate + Fe2+
MTA
CoA-SH
Nitrilase
=NIT4
Asparagine + cystine
+ NH3
Proteins
synthesis
-glutamylTranspeptidase
=-GT
-glutamyl-cyanoalanine
Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
pdxK /SOS4
PM
PMP
Transaminase
N
reduction/as
similation
pdxH/
PDX3
Glutamate
Phosphatase
pdxK /SOS4
PL
PLP
Phosphatase
Photosynthesis
DHAP RU5P
pdxH/
PDX3
Phosphatase
PN
PNP
Glycolysis
AVG
Methionine
Indole-3-acetaldehyde
oxidase
Indole-3-acetic acid
(IAA)
N metabolism
AVG targets
= Sub group I of
aminotransferase
SAM Synthase
Amino acid
+
organic acid
S-Adenosyl-L-methionine
AVG
ACC Synthase
PLP
ACC
ACC oxidase
AVG
Transaminase
PLP
Indole-3-pyruvic acid
decarboxylase
Assumed PLP enzyme
Indole-3-acetaldehyde
(IAAld)
Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
Ethylene pathway
Tryptophan
aminotransferase
PLP
Indole-3-pyruvic acid
(IPyA)
AVG
pdxK /SOS4
Nucleic acids
Tryptophan
Reductase
PDX1/PDX2
Amino acid
+
organic acid
Ethylene
tal research, recent results have shown that some of them are
not specific to the ethylene receptors.
Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
the mutants of ethylene signalling and ethylene/auxin interaction on root development. For example, co-treatment with
Co2+, AIB and 1-MCP in the presence or absence of ACC could
allow study of the signalling effect of ACC and identify the
specific protein or receptor involved.
SIDE EFFECTS OF SOME PRODUCTS AND BYPRODUCTS OF BIOSYNTHETIC PATHWAYS OF
ETHYLENE AND POLYAMINES ON ROOT
MORPHOGENESIS
Effect of competitive biosynthesis between ethylene and
polyamines on root growth
Biosynthetic pathways of ethylene and polyamines are linked
through S-adenosylmethionine via S-adenosylmethionine
decarboxylase (SAMDC) and ACS (Fig. 1A), and ethylene and
polyamines are known to have opposite effects on senescence
and fruit ripening processes (Tassoni et al. 2006; Harpaz-Saad
et al. 2012). Other interesting results come from the inhibition
of SAMDC and the ethylene biosynthetic pathway that have
been reported to reverse the inhibitory effects of ethylene on
root growth (Locke et al. 2000; Tassoni et al. 2000; Hummel
et al. 2002). Micromolar exogeneous supply of putrescine, spermine or spermidine stimulate elongation of barley roots (Locke
et al. 2000). In Arabidopsis simple and double mutant bud2-1
(SAMDC4) and samdc1 (SAMDC1) increase root proliferation
and growth through an altered homeostasis of polyamines
(Kumar et al. 1996; Ge et al. 2006). In Arabidopsis SAMDC
genes belong to a small family of four genes that are not functionally redundant. Thus alteration of the SMDC4 gene affects
growth and development, whereas alteration of the SMDC1
gene displays a more severe phenotype (Ge et al. 2006). The
double mutant SMDC4-SMDC1 (bud2-1-samdc1) induced
embryo lethality, probably through spermidine deficiency (Imai
et al. 2004). Further analyses would be helpful to understand
the partitioning flows between ACC and decarboxylated S-adenosylmethionine through the regulation ACS and SAMDC
genes and proteins. In tomato pericarp slices, putrescine, spermine and spermidine inhibit ethylene production by inhibiting
vacuolar transport of the ethylene precursor ACC. Conversely,
competitive and irreversible inhibitors of polyamine biosynthesis, such as D-arginine, L-canavanine, a-methylornithine and adifluoromethylornithine, stimulate AIB/ACC uptake into the
vacuole and ethylene production (Saftner 1989). Furthermore,
the polyamine inhibition of ethylene production can be reduced
or reversed by high concentrations of Ca2+ (Saftner 1989).
Cyanide activates ethylene receptors and inhibits root hair
development and elongation
Cyanide (HCN) and compounds like isocyanides, such as
benzyl isocyanide and cyclohexyl isocyanide (p-acceptor
compounds), are ethylene agonists able to mimic ethylene
action in the presence of the copper co-factor by eliciting inactivation of the ethylene receptors (Quinn & Yang 1989; Sisler &
Serek 1997). Recent in vitro studies have shown that ethylene
and cyanide were able to turn off autophosphorylation of the
ETR1 receptor needed for receptor inactivation. The recovery of
ethylene and cyanide-induced inhibition of ETR1 autophosphorylation by 1-MCP treatment indicated that both compounds
compete for the same binding site in the sensor domain of the
receptor (Voet-van-Vormizeele & Groth 2008). In fact, complementary in vivo and in vitro studies demonstrated that cyanide
and ethylene in the presence of the copper co-factor increase the
affinity of the receptor for the protein EIN2 (ethylene insensitive
2) by stabilizing the ETR1-EIN2 complex and allow the release
of Raf-like protein kinase CTR1 involved in the signalling cascade. A model has been proposed in which binding or release of
CTR1 protein kinase is dependent on autophosphorylation of
the ethylene receptor controlled by the binding of ethylene or
cyanide (Bisson & Groth 2010). Besides its role as an ethylene
agonist on the ethylene receptor, cyanide accumulation strongly
inhibits primary root elongation and root hair growth (Garca
et al. 2010). Indeed, mutants of mitochondrial b-cyanoalanine
synthetase (CYS-C1) obtained by insertion of a T-DNA showed
important defects in root hair formation (Fig. 1). In wild-type
plants, these root hair defects can be obtained by exogenous supply of cyanide in the growth medium. Moreover, transcriptional
profiling of the cys-c1 mutants revealed that cyanide accumulation induced repression of genes encoding enzymes involved in
cell wall rebuilding as well as genes involved in ethylene signalling and metabolism (Garca et al. 2010).
Pharmacological supply of ACC and regulation of its
endogenous levels
Generally, ACC is used as a pharmacological activator of ethylene production in agar Petri dishes in order to modulate primary root and root hair elongation (Masucci & Schiefelbein
1996; Swarup et al. 2007). However, direct supply of ACC to
the growth medium induces a shortcut in ACC biosynthesis
regulation by ACS and probably impairs or induces different
compensatory systems to regulate ethylene production. Indeed,
in Arabidopsis, it has been demonstrated that the control of circadian rhythm in ethylene production is predominantly regulated via the ACS transcript levels rather than ACO levels
(Thain et al. 2004). Moreover, permanent and high concentrations of ACC dampen the amplitude of the circadian rhythm of
ethylene production (Thain et al. 2004). A first point of regulation is also ACC transport across the plasma membrane. As
recently demonstrated, the lysine histidine transporter 1
(LHT1) is responsible of ACC uptake because the are2 mutant
(ACC-resistant 2) defective in LHT1 displays a dose-dependent
response to exogenous supply of ACC (Shin et al. 2014). Likewise, competitive inhibition of ACC transport by LHT1 with
neutral amino acids such as alanine and glycine induced suppression of the ACC-induced triple response (Shin et al. 2014).
Moreover, plants have developed different strategies for buffering excess ACC synthesis and to reduce ethylene production.
Indeed, ACC can be translocated long distances through the
xylem (Bradford & Yang 1980; Finlayson et al. 1999) and
phloem (Voesenek et al. 1990). ACC is also regulated by intracellular compartmentalisation through its utilisation, conjugation and storage into the vacuole. Within vegetative and fruit
tissues, ACC levels are regulated by conjugation into MACC,
GACC and JA-ACC, respectively (Amrhein et al. 1981; Martin
et al. 1995; Staswick & Tiryaki 2004). Thus, in Chenopodium
rubrum seedling shoots, MACC levels are about four times as
high as those of ACC and fluctuate diurnally in a similar manner (Machackova et al. 1997). Although some physiological
studies have shown that distinct carriers are involved in ACC
Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
and MACC transport into the vacuole, the identification, characterisation and regulation of genes encoding these transporters are not yet achieved (Bouzayen et al. 1989; Tophof
et al. 1989; Saftner & Martin 1993; Shin et al. 2014). Therefore,
it can be assumed that conjugation of ACC, like many hormones, can be used either to deplete the ACC pool through
long-distances transport, conjugation, storage and catabolism
in order to reduce ethylene production (Jiao et al. 1986) or to
modulate the possible signalling effects of ACC on root growth
or fruit ripening processes, as observed for IAA conjugates
(Staswick et al. 2005; Ludwig-M
uller 2011). Furthermore, in
Arabidopsis, the recent discovery of a gene encoding a homologue of the bacterial ACC deaminase (ACD) that converts
ACC to a-ketobutyrate and ammonia suggests that a catabolic
pathway for ACC exists in plants and could also be involved in
the regulation of ACC levels (McDonnell et al. 2009).
THE AMINO ACID NATURE OF ACC OPENS THE
POSSIBILITY OF ITS INVOLVEMENT AS MODULATOR
OR LIGAND ON GLUTAMATE RECEPTORS
Because plant glutamate receptors (GLR), mainly located in the
roots, can be gated by at least 12 proteinogenic amino acids
(Vincill et al. 2012, 2013; Tapken et al. 2013), the non-proteinogenic amino acid character of ACC raises questions about
ACC-GLR interactions on root morphogenesis.
Plants glutamate receptors: a perspective from animal science
In Arabidopsis, 20 GLR genes have been identified and are
highly expressed in primary and lateral roots (Chiu et al. 2002;
Price et al. 2012). The structure of GLRs is organised into four
different units (Fig. 3) composed by an amino terminal
domain (ATD), a ligand binding domain (LBD), a trans-membrane domain (TMD) formed by three complete trans-membrane domains (M1, M3 and M4) with one re-entrant loop
(M2) that forms the ion channel (similar to an inverted K+
channel) and a C-terminal tail involved in coupling with cytoplasmic signalling pathways. As in mammals, GLRs most probA
GLR1.4
GLR3.4
NH2
Unknown
modulators
ATD
NR2B
ATD
ATD
LBD
LBD
Polyamines:
Spermine,
Spermidine,
Antagonists
Arg
Agonists
Met>Trp>Phe>
Leu>Tyr>Asn
>Thr
LBD
Out
COOH
Ca2+, Na+, K+
In
Agonists
Glutamate
Out
COOH
Ca2+
Agonists
Glycine,
H2S
ACC
AIB?
M4
M1
M4
M1
In
Antagonists
Agonists
Asn>Ser>Gly
M3
Out
M3
TMD
NR1
NH2
In
Ca2+
Fig. 3. Schematic of glutamate receptors in plants (GLR) and animals (iGLR). A: The ligands acting on Arabidopsis receptors AtGLR1.4 and AtGLR3.1 are presented. Although the full receptor is a tetramer, only a monomer of AtGLR1.4 and AtGLR3.1 is presented. M1M4 refer to the transmembrane domains in the
iGluR/GLR family. B: Hypothetical mechanism of potentiation of iGLRs in mammals by allosteric regulation of polyamines after dimerisation of the receptor subunits. The polyamine molecules can directly bind and stabilize the amino-terminal domain (ATD) or the ligand binding domain (LBD) dimer interface. TMD,
transmembrane domain.
Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
nes, methionine and arginine are, respectively, the best agonist and antagonist of plant AtGLR1.4 and play major roles
in promoting or inhibiting root initiation and elongation
(Tapken et al. 2013). This could confirm the recent hypothesis that ACC could act as a signal molecule (Yoon & Kieber
2013; Van de Poel & Van Der Straeten 2014).
Could polyamines, HCN and H2S bind to plant glutamate
receptors as modulators?
In mammals, the amino terminal domains (ATD) of iGLRs
mediate allosteric effects by directly binding exogenous modulators such as H+, Mg2+ and Zn2+ ions, polyamines, neurosteroids and drugs. Although GLRs have a similar ATD domain,
no study has yet been conducted in plants to highlight positive
or negative allosteric modulators. Intriguingly, it has been
shown that cyanide concentrations as low as 10 lM can act as a
potent neuromodulator of iGluRs of the central nervous system
and enhances accumulation of Ca2+ in rat cerebellar granule
cells (Borowitz et al. 1997; Sun et al. 1999). In addition, cyanide degradation by b-cyanoalanine synthetase produces
hydrogen sulphide (H2S), another gaseous compound that
enhances NMDA receptor-mediated responses and facilitates
the induction of their long-term potentiation in mammals
(Kimura 2013). Furthermore, polyamines such as spermine
and spermidine are also known as allosteric modulators of
iGluR receptors by acting on specific sites located under the
ATD (Mony et al. 2009). Mechanisms by which polyamines
enhance activity of receptor are not clear (Fig. 3B), but it is
assumed that polyamines would bind at the LBD dimer interface and stabilise dimmer assembly in the tetramer or would
bind at the level of ATD by gluing together these lobes.
Because all these molecules in plants are produced upstream
from ethylene perception, further investigations on their effective role in root growth in relation to ethylene effects are
required. This would allow a reconsideration of previous studies of ethylene signalling effects based on pharmacological
treatments. For example, calcium requirements for a variety of
ethylene-dependent processes such as pathogenesis-related
responses and the triple response (Raz & Fluhr 1992) could be
reconsidered from this viewpoint.
SIDE EFFECTS OF ETHYLENE INHIBITORS CAN BE AN
OPPORTUNITY TO FIND NEW TARGETS INVOLVED IN
THE ROOT MORPHOGENETIC PROGRAMME
Control of N and C metabolism in the biosynthesis of
ethylene and auxin during root morphogenesis
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