Plant Biology ISSN 1435-8603

REVIEW ARTICLE

Non-specificity of ethylene inhibitors: double-edged tools to

find out new targets involved in the root morphogenetic
programme
E. Le Deunff1,2 & J. Lecourt3
1 Normandie Universit
e, UMR EVA, F-14032, Caen cedex, France
2 INRA, UMR 950,

Ecophysiologie V

eg

etale & Agronomie, Nutritions NCS, INRA, F-14032 Caen cedex, France
3 East Malling Research, East Malling, Kent, UK

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
d
Ecophysiologie 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.

Received: 21 September 2015; Accepted: 24

September 2015
doi:10.1111/plb.12405

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-

lene precursor 1-aminocyclopropane-1-carboxylic acid (ACC)

inhibit the primary root elongation as quickly as auxin
(Jackson 1991; Le et al. 2001), combining genetic and pharmacological approaches have been used to obtain more insight
into the ethylene-induced root inhibition. The manipulation of
growth of the exploratory root and root hair systems can easily
be achieved by pharmacological treatments that inhibit or activate ethylene production and perception (Dugardeyn & Van
Der Straeten 2008; Grierson et al. 2014). Since the 1970s, several pharmacological inhibitors of the two enzymes involved in
ethylene biosynthesis, ACC synthase (ACS) and ACC oxidase
(ACO), have been found and intensively used. Thus, L-a-(2aminoethoxyvinyl)glycine (AVG) and 2-(aminooxy)acetic acid
(AOA) are mainly used to inhibit ACS activity, whereas cyclopropane-carboxylic acid (CCA) and a-aminoisobutyric acid
(AIB) are used to inhibit ACO. Likewise, silver nitrate
(AgNO3), silver thiosulphate (Ag(S2O3)23
), 2,5 norbornadiene and 1-methylcyclopropene (1-MCP) are inhibitors of ethylene perception involved at ethylene receptor level. The
biochemical effects of these compounds were discovered thanks
to their inhibitory action on ethylene production and perception in fruit slices, flowers and vegetative tissues such as roots
(Apelbaum et al. 1981; Broun & Mayak 1981; Jackson 1991).

Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands

Ethylene inhibitors: double-edged tools

Le Deunff & Lecourt

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 carbonmg
1
root DW) in seedlings, we still do not know how ethylene and
urea

Ornithine

auxin biosynthesis are coordinated with C and N metabolism

during the root morphogenetic programming. Recent results
highlight that the daily root elongation during the increasing of
ACC concentrations is correlated with seedlings C and N status
as well as with the root aspartate content (Lemaire et al. 2013).
Because aspartate is known to play a central role in the C/N
balance (Coruzzi 2003) and auxin catabolism (Ludwig-M
uller
2011), this raises the possibility of uncovering interactions
between primary metabolism and ethylene signalling.
In this review, we focus on the modulation of root growth
by pharmacological activation or inhibition of the ethylene
biosynthesis pathway and less on ethylene perception. We
highlight the pitfalls of the pharmacological approach used to
modulate root and root hair growth, but also show how this
approach can revise our appreciation of ethylene biosynthesis
and perception inhibitors, and can assist us in finding major
new targets involved in the morphogenetic root programme.
PHARMACOLOGICAL MANIPULATIONS OF ETHYLENE
BIOSYNTHESIS: A DEVIOUS TRAP
Ethylene biosynthesis pathway
Ethylene is derived from methionine that is converted into
S-adenosyl-methionine (SAM) by the SAM synthetase enzyme
(Yang & Hoffman 1984). SAM is then converted into ACC
(1-aminocyclopropane-1-carboxylic acid), the ethylene precursor, and MTA (50 -methylthioadenosine). This step is catalysed
by ACC synthase (ACS), an aminotransferase requiring pyridoxal-50 -phosphate as a cofactor. In a second step, ACC is
oxidised in the presence of ascorbate by an O2-activating nonheme iron enzyme called ACC oxidase (ACO) containing a single ferrous ion. The ACO enzyme produces stochiometrically:
ethylene, dehydroascorbate (DHA), CO2 and hydrogen cyanide
(HCN), as presented in Fig. 1 (Dong et al. 1992).
The ACC can also be diverted by conjugation with malonylCoA to form 1-(malonylamino)-ACC (MACC), with glu-

CO2

Spermidine

Putrescine

Arginine

Spermine

H2O
CO2 + NH3

Agmatine

MTA

dSAM

MTA

CO2

Sam dcarboxylase
= SamDc
Proteins
synthesis

CO2

AIB, CCA, Co2+

AVG, AOA
ACC synthase=ACS
S-AdoMe

ATP

PPi + Pi

Methionine
2-oxo-acid

ACC oxidase= ACO

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

Ethylene + CO2 + HCN + DHA

O2 + ascorbate + Fe2+

MTA

CoA-SH

Nitrilase
=NIT4

Asparagine + cystine
+ NH3

Proteins
synthesis

-glutamylTranspeptidase
=-GT
-glutamyl-cyanoalanine

Fig. 1. Pathways of ethylene synthesis and its linkage to

polyamine biosynthesis and hydrogen cyanide catabolism in higher plants. HCN, hydrogen cyanide; dSAM,
decarboxylated S-adenosylmethionine; ACC, 1-aminocyclopropane carboxylic acid; S-AdoMe, S-adenosylmethionine; GACC, N-glutamyl-ACC; MACC, Nmalonyl-ACC; GSH, reduced glutathione; DHA, dehydroascorbate; MTA, 50 -m
ethylthio-ad
enosine; MTR, 5methylthioribose; MTR-1-P, 5-methylthioribose-1-P;
KMB, 2-keto-4-methylthio-butyric acid; PLP, pyridoxal
phosphate. In red are molecules known to be involved as
agonists of the iGLuRs/GLRs, in blue are antagonists and
in green are iGLuR modulators. AVG, L-a-(2-aminoethoxyvinyl)glycine and AOA, aminooxy-acetic acid
are inhibitors of the ACS enzyme. AIB, a-aminoisobutyric
acid and CCA, cyclopropanecarboxylic acid are inhibitors
of the ACO enzyme.

Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands

Le Deunff & Lecourt

Ethylene inhibitors: double-edged tools

tathione to form 1-(c-L-glutamylamino)-ACC (GACC) and

with jasmonic acid (JA) to form jasmonyl-ACC (JA-AAC)
through the action of an ACC N-malonyltransferase, a c-glutamyltranspeptidase and a JA-amino synthetase, respectively
(Yang & Hoffman 1984; Martin et al. 1995; Staswick & Tiryaki
2004).
The poisonous gas HCN is detoxified via b-cyanoalanine synthase (CAS) and nitrilase 4 (NIT4); CAS converts HCN in the
presence of L-cysteine to b-cyanoalanine and hydrogen sulphide
(H2S) (Fig. 1). Then, NIT4 catalyses the conversion of
b-cyanoalanine to aspartate, asparagine and ammonia, which are
typical compounds used in N metabolism (Piotrowski 2008).

2010). For instance, AVG strongly inhibits auxin biosynthesis

activity of the PLP-dependent tryptophan aminotransferase
enzyme (Soeno et al. 2010). These results confirm previous studies (Wagner et al. 2006; Raschke et al. 2011) conducted on pyridoxine biosynthesis genes (PDX) involved in PLP (vitamin B6)
biosynthesis in Arabidopsis (Fig. 2A). Indeed, pdx1.1 and pdx1.3
knockout mutants impaired root growth, reduced meristem size
and altered root cell division and elongation (Chen & Xiong
2005; Titiz et al. 2006). Recent genetic analyses have demonstrated that the pdx1.3 mutant is defective in both conversion of
anthranilate to tryptophan (Trp) and Trp to auxin in roots
(Chen & Xiong 2009a,b). Furthermore, root growth impairment
in the pdx1.3 mutant is propagated by a deficit in ethylene production that induces an auxin gradient deficit in the root tip,
leading to reduced cell division and expansion (Chen & Xiong
2009a; Boycheva et al. 2015).
The inhibitory action of AVG on aminotransferases involved
in N metabolism has been demonstrated in Brassica napus
seedlings. AVG treatment significantly increased the root and
shoot concentrations of free amino acids, such as asparagine,
aspartic acid, glutamine and glutamic acid, demonstrating that
some aminotransferases implicated in N metabolism are also
impaired by AVG treatment (Lemaire et al. 2013). This effect
has also been demonstrated through the restoration of elongation in the exploratory root system after addition of 1 mM
potassium glutamate to AVG-treated roots (Leblanc et al.
2008). Accordingly, because of the multiple targets of AVG (see
below), conclusions drawn from pharmacological studies using
AVG as a specific inhibitor of ACS should be taken with
caution (Fig. 2B).

Some problems linked to inhibition of the ACS enzyme

The most efficient inhibitor of the ACS enzyme, L-a-(2-aminoethoxyvinyl)glycine (AVG), belongs to a family of olefinic
glycine analogues (Lieberman 1979; Satoh & Yang 1989). This
family of compounds acts as terminal inhibitors of many
enzymes of the aminotransferase subgroup I that contains ACC,
aspartate, alanine, tyrosine, histidine-phosphate and phenylalanine amino-transferases (Christen & Mehta 2001; Liepman &
Olsen 2004). As a result, doubt arises in the specificity of this
family of compounds (Fig. 2), as they can inhibit both transaminases involved in IAA biosynthesis (Soeno et al. 2010) as well as
those involved in N metabolism (Leblanc et al. 2008; Lemaire
et al. 2013). The recent use of chemical genomic-based strategies
has allowed the identification and characterisation of chemical
compounds acting as highly specific inhibitors of enzymes of
ethylene and auxin biosynthesis (Lin et al. 2010; Soeno et al.
A

pdxK /SOS4

PM

PMP
Transaminase

N
reduction/as
similation

pdxH/
PDX3

Glutamate

Phosphatase

pdxK /SOS4

PL

PLP

Glutamine + G3P + R5P

Phosphatase

Photosynthesis

DHAP RU5P

pdxH/
PDX3

Triose phosphate/Pentose phosphate pool

Phosphatase

PN

PNP
Glycolysis

AIA IPyA pathway

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

Fig. 2. Importance of PLP-dependent enzymes in the

metabolic pathways of ethylene, auxin and N. A: De
novo biosynthesis and salvage pathways of PLP in plants.
The different chemical forms of vitamin B6 obtained by
inter-conversion in the salvage pathway correspond to
PN (pyridoxine), PL (pyridoxal), PM (pyridoxamine) and
the phosphorylated forms to PNP, PLP and PMP respectively. Thin arrows represent the enzymes not yet identified in Arabidopsis (adapted from Raschke et al. 2011).
B: Targets of AVG inhibitor on PLP-dependent enzymes
involved in ethylene and IAA biosynthesis pathways. The
amino-transferase subgroup I contains ACC, aspartate,
alanine, tyrosine, histidine-phosphate and phenylalanine
amino-transferases. G3P = glyceraldehyde-3-phosphate;
DHAP = dihydroacetone phosphate; R5P = ribose-5phosphate; RU5P = ribulose-5-phosphate.

Reductase

PDX1/PDX2

Amino acid
+
organic acid

Ethylene

Ethylene inhibitors: double-edged tools

Le Deunff & Lecourt

tal research, recent results have shown that some of them are
not specific to the ethylene receptors.

Some problems linked to inhibition of the ACO enzyme

Several inhibitors of the ACO enzyme have been identified
(Fig. 1). Some of them are competitive inhibitors of ACO acting as structural analogues of ACC such as cyclopropanecarboxylic acid (CCA) and a-aminoisobutyric acid (AIB)
(Apelbaum et al. 1981; Satoh & Esashi 1983; Brunhuber et al.
2000). The Ki of CCA (7.6  0.6 mM) is four times higher than
that of AIB (2  0.1 mM), and few studies have use CCA as
inhibitor on the ACO enzyme (Apelbaum et al. 1981; Brunhuber et al. 2000). AIB is the most used competitive inhibitor of
ACO and the most intriguing (Satoh & Esashi 1983). Indeed,
AIB is also a substrate of ACO producing acetone, ammonia
and CO2 without HCN production (Charng et al. 1997). The
Ki of AIB is nearly 30-fold higher than the Km (62  4 lM) of
ACC (Brunhuber et al. 2000). Accordingly, millimolar concentrations of AIB (15 mM) are required to inhibit ethylene production and to restore hypocotyl or primary root elongation
(Guzman & Ecker 1990; Xu et al. 2008; Tsang et al. 2011).
However, when AIB is used in micromolar concentrations
(0.510 lM), it is also able to enhance root elongation (Leblanc
et al. 2008) similarly to low AVG concentrations (5 lM) (Le
et al. 2001). Because AIB is also known as a competitive inhibitor of ACC, ACC tonoplast transporter and glutamine membrane transporter, it likely modifies ACC homeostasis through
its utilisation, conjugation and vacuolar storage (Bennett &
Spanswick 1983; Martin et al. 1995).
It is reported that AIB cancels the cell wall stress induced by
isoxaben treatment (a cellulose-damaging agent) and restores
elongation of the primary root cells. It is assumed that AIB
competes with ACC on an early ACC-dependent signalling
pathway induced independently of ethylene perception during
the cell wall response (Tsang et al. 2011). These results do not
exclude that AIB could act on other targets or counteract ACC
binding to a specific protein involved in the signalling pathway
of root elongation.
Ions have also been reported to act as inhibitors of ACO:
Co2+ and Ni2+ compete with Fe2+ for the active site in ACO
during ethylene production and can reduce irreversibly the
ethylene biosynthesis when used at micromolar (1030 lM)
concentrations (Yang & Hoffman 1984; Romera et al. 1996;
Brunhuber et al. 2000). Metal chelating agents such as 1,10
phenanthroline (PA), 1,2 dihydroxynapthalene and
diethyldithiocarbamate (DIECA) have been previously found
to inhibit ethylene production, providing the first evidence that
iron is an essential cofactor for the conversion of ACC into
ethylene by ACO (Bouzayen et al. 1991; Hamilton et al. 1991).
Insofar as Co2+ probably inhibits many other enzymes that
require Fe2+ or not for their catalytic activity (Palit et al. 1994),
co-treatments with Co2+ and AIB would allow us to reduce
concentrations of these inhibitors and their side effects in order
to explore new signalling pathway in root elongation.
PHARMACOLOGICAL MANIPULATIONS OF ETHYLENE
PERCEPTION
Four pharmacological agents have been discovered and used
successively: silver ion (Ag+), applied as silver nitrate (AgNO3)
or silver thiosulphate (Ag(S2O3)23
), and the synthetic gaseous
compounds 2,5 norbornadiene and 1-methylcyclopropene
(1-MCP). Although these inhibitors are still used in fundamen4

Silver ion promotes an auxin efflux independently of its

effects on ethylene receptors
The silver ion (Ag+), applied as silver nitrate (AgNO3) or silver
thiosulphate (Ag(S2O3)23
), was the first inhibitor used to
block ethylene perception (Burg & Burg 1967; Beyer 1976). It is
assumed that Ag+ ions bind to the ethylene receptors at the
same site normally occupied by copper (Guzman & Ecker
1990; Zhao et al. 2002; Binder et al. 2010). However, in Arabidopsis recent results indicated that among the five isoforms of
ethylene receptors (ETR1, ESR1, ETR2, ESR2, EIN4) silver supports ethylene binding only to ETR1 and ESR1 isoforms. The
effects of silver are mostly dependent upon ETR1, and silver
may have an effect on receptors outside of the copper binding
location, leading to a reduced number of ethylene binding sites
generated by copper (McDaniel & Binder 2012). Furthermore,
despite compelling evidence that treatments with micromolar
concentrations of AgNO3 (10100 lM) can restore elongation
of primary root and hypocotyl in the ethylene-overproducing
mutant eto1-1 (Guzman & Ecker 1990), recent results suggest
that restoration of root elongation is caused by the promotion
of IAA root efflux independently of the effect of Ag+ on ethylene perception (Strader et al. 2009). Thus, the reduction of
root cell expansion in eto1 seedlings was fully suppressed by
blocking auxin influx with aux1 mutation and partially
impeded by dampening auxin signalling (Strader et al. 2010).
This result is consistent with the absence of root apoplast acidification in the aux1 mutant treated with ACC compared to
ACC control seedlings (Staal et al. 2011), suggesting that control of the active state of plasma membrane H+-ATPases is one
of the mechanisms by which ethylene, via auxin transport and
signalling, affects root cell expansion. In this respect, the mild
effects on exploratory root elongation observed after AVG
treatment is also consistent with inhibition effects of AVG on
ethylene and auxin biosynthesis enzymes (Leblanc et al. 2008;
Soeno et al. 2010).
1-Methylcyclopropene is the most powerful and effective
inhibitor of ethylene receptors
Among the unsaturated cyclic olefins family, the synthetic gaseous compound 1-methylcyclopropene (1-MCP) acts at very low
concentrations as a competitive inhibitor of ethylene receptors by
blocking them irreversibly (Sisler & Serek 1997). The affinity of
1-MCP for the ethylene receptors is ten times higher than that
ethylene, and the concentration of 1-MCP used is around one
order of magnitude lower than other used inhibitors such as 2,5
norbornadiene (Sisler & Pian 1973) and trans-cyclooctene (Sisler
et al. 1990). In addition to its role as potent inhibitor of ethyleneinduced triple response in dark-grown Arabidopsis seedlings, 1MCP has allowed the characterisation of ethylene binding sites of
ERS1 and ETR1 ethylene receptors in heterologous systems such
as yeast (Hall et al. 2000). Although the use of 1-MCP in a Petri
dish under a flowing system is delicate, manipulating a commercial powder product such as Ethylbloc (Floralife) is easier to study
its effects on root development in agar plates (Xu et al. 2008).
In summary, cross-combination experiments with multiple,
diversely acting chemicals would help to validate the effects of

Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands

Le Deunff & Lecourt

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

Ethylene inhibitors: double-edged tools

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 (Garc
a
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 (Garc
a 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

Ethylene inhibitors: double-edged tools

Le Deunff & Lecourt

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

ably form tetrameric channels and are constitutively active

Ca2+-permeable non-selective cation channels (Tapken & Hollmann 2008). In contrast to the ionotropic glutamate receptor
(iGLR) counterpart, GLRs can be gated by at least 12 proteinogenic amino acids (Fig. 3A) as well as by an unknown number
of small molecules and peptides such as GSH (Forde & Roberts
2014). Indeed, patch-clamp studies in heterologous expression
of plant AtGLR3.2 and AtGLR3.4 in transfected human embryonic kidney (HEK) cells has shown that Asn, Ser and Gly, but
not glutamate, are the primary ligand-gated channels of GLR
(Vincill et al. 2012, 2013). Likewise, a study using heterologous
expression of AtGLR1.4 in Xenopous oocytes demonstrated that
AtGLR1.4 functioned as a ligand-gated, non-selective, Ca2+permeable cation channel (Tapken et al. 2013). Among the 20
natural amino acids tested, AtGLR1.4 was activated by seven
amino acids. Methionine was the most effective, followed by
tryptophan, phenylalanine, leucine, tyrosine asparagine and
threonine. Arginine was the most effective antagonist amino
acid of the methionine action.
Does ACC binds to plant glutamate receptors as a modulator
or as a ligand?
In plants, ACC is a neutral and non-proteinogenic amino
acid that has a chemical formula and molecular mass close
to c-aminobutyric acid (GABA) and alpha-aminoisobutyric
acid (AIB) while in mammals, ACC is a synthetic molecule
called ACPC and used as a partial agonist of ionotropic glutamate receptor (Fig. 3B) on glycine binding sites (NahumLevy et al. 1999; Inanobe et al. 2005). Thus, ACC-ACPC is
able to induce 80% of the activity of iGluRs (Inanobe et al.
2005). Because of the importance of ethylene in root elongation (R
uzicka et al. 2007; Swarup et al. 2007), the strong
expression of plant glutamate receptor-like genes (GLRs) in
exploratory and root hair systems (Chiu et al. 2002) and the
role of GLR in lateral root initiation (Vincill et al. 2013),
ACC is likely to be a ligand of plant GLRs on the ligand
binding domain. Indeed, it is very intriguing that the two
precursors of biosynthetic pathways of ethylene and polyamiB

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

Le Deunff & Lecourt

Ethylene inhibitors: double-edged tools

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
Pharmacological and genomic-based approaches have highlighted the non-specificity of AVG suicide PLP enzyme inhibiREFERENCES
Amrhein N., Schneebeck D., Skorupka H., Tophof S.,
St
ockigt J. (1981) Identification of a major metabolite of the ethylene precursor 1-aminocyclopropane1-carboxylic acid in higher plants. Naturwissenschaften, 68, 619620.
Apelbaum A., Wang S.Y., Burgoon A.C., Baker J.E.,
Lieberman M. (1981) Inhibition of the conversion of
1-aminocyclopropane-1-carboxylic acid to ethylene

tor, commonly used to block ACS enzyme activity in order to

inhibit primary root elongation and root hair formation in
Arabidopsis and B. napus seedlings (Masucci & Schiefelbein
1996; Leblanc et al. 2008; Soeno et al. 2010). Indeed, the
screening of inhibitors showing chemical analogy to AVG has
shown that AVG and these compounds exhibit anti-auxin
activity (Soeno et al. 2010). AVG treatment (10 lM) significantly impairs concentrations of all free amino acids in shoots
and roots compared to control seedlings (Lemaire et al. 2013),
and co-treatment with 1 mM potassium glutamate can lead to
an almost complete restoration of elongation of the exploratory
root system (Leblanc et al. 2008).
These studies also reveal the importance of pyridoxal-50 phosphate (PLP), an active form of vitamin B6, as cofactor for
enzymes involved in N metabolism and therefore the biosynthesis of ethylene and auxin that are produced from both the
amino acids methionine and tryptophan, respectively (Christen
& Mehta 2001). In addition, they corroborate different studies
performed with mutants of PLP biosynthesis enzymes that have
an effect on root growth (Chen & Xiong 2005; Titiz et al. 2006;
Boycheva et al. 2015). Together, these results lead to the
assumption that the PLP cofactor represents a metabolic hub
between N and C metabolism and biosynthesis of ethylene and
auxin (Fig. 2). This interconnection highlights the importance
PLP-dependent enzymes in the root morphogenetic programme and offers the possibility in future to modulate N use
efficiency through targeted mutations or the use of more specific PLP enzyme inhibitors.
CONCLUDING REMARKS
Some of the pharmacological molecules used to inhibit ethylene biosynthesis and perception are not sufficiently reliable to
complete and validate genetic works on the effects of ethylene
and ethylene/auxin interactions on root growth. Indeed,
manipulating the ethylene biosynthetic pathway to modulate
root system architecture requires keeping in mind the potential side effects of inhibitors on root growth via impairment by
N and C primary metabolism, Ca2+ signalling and modulation
of IAA biosynthesis and signalling. In contrast, these pharmacological inhibitors can be essential to highlight the interconnections in root developmental responses between hormonal
pathways and metabolism of N and C. In this respect, genomic
chemistry or a systems genomic approach can provide complementary tools to pinpoint major targets or central hub(s) in
primary metabolism involved in regulation of the root morphogenetic programme. Identification of such regulatory hub
(s) is essential to our knowledge of the root morphogenetic
programme and structurefunction relationships in order to
improve the efficiency of macronutrient absorption.

by structural analogs, inhibitors of electron transfer,

uncouplers of oxidative phosphorylation, and free
radical scavengers. Plant Physiology, 67, 7479.
Bennett A.B., Spanswick R.M. (1983) Derepression of
amino acid-H+ cotransport in developing soybean
embryos. Plant Physiology, 72, 781786.
Beyer E.M. (1976) A potent inhibitor of ethylene action
in plants. Plant Physiology, 58, 268271.
Binder B.M., Rodr
guez F.I., Bleecker A.B. (2010) The
copper transporter RAN1 is essential for biogenesis

Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands

of ethylene receptors in Arabidopsis. Journal of Biological Chemistry, 285, 3726337270.

Bisson M.M., Groth G. (2010) New insight in ethylene
signaling: autokinase activity of ETR1 modulates the
interaction of receptors and EIN2. Molecular Plant,
3, 882889.
Borowitz J.L., Gunasekar P.G., Isom G.E. (1997)
Hydrogen cyanide generation by l-opiate receptor
activation: possible neuromodulatory role of
endogenous cyanide. Brain Research, 768, 294300.

Ethylene inhibitors: double-edged tools

Bouzayen M., Latch
e A., Pech J.-C., Marigo G. (1989)

Carrier-mediated uptake of 1-(malonylamino)
cyclopropane-1-carboxylic acid in vacuoles isolated
from Catharanthus roseus cells. Plant Physiology, 91,
13171322.
Bouzayen M., Felix G., Latch
e A., Pech J.-C., Boller T.
(1991) Iron: an essential cofactor for the conversion
of 1-aminocyclopropane-1-carboxylic acid to ethylene. Planta, 184, 244247.
Boycheva S., Dominguez A., Rolcik J., Boller T., Fitzpatrick T.B. (2015) Consequences of a deficit in vitamin B6 biosynthesis de novo for hormone
homeostasis and root development in Arabidopsis.
Plant Physiology, 167, 102117.
Bradford K.J., Yang S.F. (1980) Xylem transport of 1aminocyclopropane-1-carboxylic acid, an ethylene
precursor, in waterlogged tomato plants. Plant Physiology, 65, 322326.
Broun R., Mayak S. (1981) Aminooxyacetic acid as an
inhibitor of ethylene synthesis and senescence in carnation flowers. Scientia Horticulturae, 15, 277282.
Brunhuber N.M., Mort J.L., Christoffersen R.E., Reich
N.O. (2000) Steady-state kinetic mechanism of
recombinant avocado ACC oxidase: initial velocity
and inhibitor studies. Biochemistry, 39, 1073010738.
Burg S.P., Burg E.A. (1967) Molecular requirements
for the biological activity of ethylene. Plant Physiology, 42, 144152.
Charng Y., Liu Y., Dong J., Yang S., Kanellis A., Kende
H., Grierson D. (1997) On 1-aminocyclopropane-1carboxylic acid oxidase. In: Kanellis A. (Ed.), Biology
and biotechnology of the plant hormone ethylene.
Kluwer Academic, Boston, MA, USA, pp 2329.
Chen H., Xiong L. (2005) Pyridoxine is required for postembryonic root development and tolerance to osmotic
and oxidative stresses. The Plant Journal, 44, 396408.
Chen H., Xiong L. (2009a) Localized auxin biosynthesis and postembryonic root development in Arabidopsis. Plant Signaling & Behavior, 4, 752754.
Chen H., Xiong L. (2009b) The short-rooted vitamin
B6-deficient mutant pdx1 has impaired local auxin
biosynthesis. Planta, 229, 13031310.
Chiu J.C., Brenner E.D., DeSalle R., Nitabach M.N.,
Holmes T.C., Coruzzi G.M. (2002) Phylogenetic and
expression analysis of the glutamate-receptorlike
gene family in Arabidopsis thaliana. Molecular Biology and Evolution, 19, 10661082.
Christen P., Mehta P.K. (2001) From cofactor to
enzymes. The molecular evolution of pyridoxal-50 phosphate-dependent enzymes. The Chemical
Record, 1, 436447.
Coruzzi G.M. (2003) Primary N-assimilation into
amino acids in Arabidopsis. The Arabidopsis Book/
American Society of Plant Biologists, 2, 117.
Dong J.G., Fernandez-Maculet J.C., Yang S.F. (1992)
Purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple fruit. Proceedings of the National Academy of Sciences USA, 89,
97899793.
Dugardeyn J., Van Der Straeten D. (2008) Ethylene:
fine-tuning plant growth and development by stimulation and inhibition of elongation. Plant Science,
175, 5970.
Finlayson S.A., Lee I.-J., Mullet J.E., Morgan P.W.
(1999) The mechanism of rhythmic ethylene production in sorghum. The role of phytochrome B and
simulated shading. Plant Physiology, 119, 10831090.
Forde B.G., Roberts M.R. (2014) Glutamate receptorlike channels in plants: a role as amino acid sensors
in plant defence? F1000prime Reports, 6, 37.

Le Deunff & Lecourt

Fraas S., Niehoff V., L

uthen H. (2014) A high-throughput imaging auxanometer for roots and hypocotyls
of Arabidopsis using a 2D skeletonizing algorithm.
Physiologia Plantarum, 151, 112118.
Garc
a I., Castellano J.M., Vioque B., Solano R., Gotor
C., Romero L.C. (2010) Mitochondrial b-cyanoalanine synthase is essential for root hair formation in
Arabidopsis thaliana. The Plant Cell Online, 22,
32683279.
Ge C., Cui X., Wang Y., Hu Y., Fu Z., Zhang D., Cheng
Z., Li J. (2006) BUD2, encoding an S-adenosylmethionine decarboxylase, is required for Arabidopsis growth and development. Cell Research, 16, 446
456.
Grierson C., Nielsen E., Ketelaarc T., Schiefelbein J.
(2014) Root hairs. The Arabidopsis Book/American
Society of Plant Biologists, 12, 125.
Guzman P., Ecker J.R. (1990) Exploiting the triple
response of Arabidopsis to identify ethylene-related
mutants. The Plant Cell Online, 2, 513523.
Hager A. (2003) Role of the plasma membrane H+ATPase in auxin-induced elongation growth: historical and new aspects. Journal of Plant Research, 116,
483505.
Hall A.E., Findell J.L., Schaller G.E., Sisler E.C.,
Bleecker A.B. (2000) Ethylene perception by the
ERS1 protein in Arabidopsis. Plant Physiology, 123,
14491458.
Hamilton A., Bouzayen M., Grierson D. (1991) Identification of a tomato gene for the ethylene-forming
enzyme by expression in yeast. Proceedings of the
National Academy of Sciences USA, 88, 74347437.
Harpaz-Saad S., Yoon G.M., Mattoo A.K., Kieber J.J.
(2012) The formation of ACC and competition
between polyamines and ethylene for SAM. Annual
Plant Reviews, The Plant Hormone Ethylene, 44, 56.
Hummel I., Cou
ee I., El Amrani A., Martin-Tanguy J.,
Hennion F. (2002) Involvement of polyamines in
root development at low temperature in the subantarctic cruciferous species Pringlea antiscorbutica.
Journal of Experimental Botany, 53, 14631473.
Imai A., Matsuyama T., Hanzawa Y., Akiyama T.,
Tamaoki M., Saji H., Shirano Y., Kato T., Hayashi
H., Shibata D. (2004) Spermidine synthase genes are
essential for survival of Arabidopsis. Plant Physiology, 13, 15651573.
Inanobe A., Furukawa H., Gouaux E. (2005) Mechanism of partial agonist action at the NR1 subunit of
NMDA receptors. Neuron, 47, 7184.
Jackson M.B. (1991) Ethylene in root growth and
development. In: Matoo A.K., Suttle J.C. (Eds), The
plant hormone ethylene. CRC Press, Boca Raton, FJ,
USA, pp 159181.
Jiao X.-Z., Philosoph-Hadas S., Su L.-Y., Yang S.F.
(1986) The conversion of 1-(malonylamino) cyclopropane-1-carboxylic acid to 1-aminocyclopropane1-carboxylic acid in plant tissues. Plant Physiology,
81, 637641.
Kimura H. (2013) Physiological role of hydrogen sulfide and polysulfide in the central nervous system.
Neurochemistry International, 63, 492497.
Kumar A., Taylor M.A., Arif S.A., Davies H.V. (1996)
Potato plants expressing antisense and sense S-adenosylmethionine decarboxylase (SAMDC) transgenes show altered levels of polyamines and ethylene:
antisense plants display abnormal phenotypes. The
Plant Journal, 9, 147158.
Le J., Vandenbussche F., Van Der Straeten D., Verbelen
J.-P. (2001) In the early response of Arabidopsis
roots to ethylene, cell elongation is up-and down-

regulated and uncoupled from differentiation. Plant

Physiology, 125, 519522.
Leblanc A., Renault H., Lecourt J., Etienne P., Deleu
C., Le Deunff E. (2008) Elongation changes of
exploratory and root hair systems induced by
aminocyclopropane carboxylic acid and aminoethoxyvinylglycine affect nitrate uptake and Bn
Nrt2.1 and BnNrt1.1 transporter gene expression in
oilseed rape. Plant Physiology, 146, 19281940.
Lemaire L., Deleu C., Le Deunff E. (2013) Modulation
of ethylene biosynthesis by ACC and AIB reveals a
structural and functional relationship between the
K15NO3 uptake rate and root absorbing surfaces.
Journal of Experimental Botany, 64, 27252737.
Lieberman M. (1979) Biosynthesis and action of ethylene. Annual Review of Plant Physiology, 30, 533591.
Liepman A.H., Olsen L.J. (2004) Genomic analysis of
aminotransferases in Arabidopsis thaliana. Critical
Reviews in Plant Sciences, 23, 7389.
Lin L.-C., Hsu J.-H., Wang L.-C. (2010) Identification
of novel inhibitors of 1-aminocyclopropane-1-carboxylic acid synthase by chemical screening in Arabidopsis thaliana. Journal of Biological Chemistry,
285, 3344533456.
Locke J.M., Bryce J.H., Morris P.C. (2000) Contrasting
effects of ethylene perception and biosynthesis inhibitors on germination and seedling growth of barley
(Hordeum vulgare L.). Journal of Experimental Botany, 51, 18431849.
Ludwig-M
uller J. (2011) Auxin conjugates: their role for
plant development and in the evolution of land plants.
Journal of Experimental Botany, 62, 17571773.
Machackova I., Chauvaux N., Dewitte W., van Onckelen H. (1997) Diurnal fluctuations in ethylene formation in Chenopodium rubrum. Plant Physiology,
113, 981985.
Martin M.N., Cohen J.D., Saftner R.A. (1995) A new 1aminocyclopropane-1-carboxylic acid-conjugating
activity in tomato fruit. Plant Physiology, 109, 917
926.
Masucci J.D., Schiefelbein J.W. (1996) Hormones act
downstream of TTG and GL2 to promote root hair
outgrowth during epidermis development in the
Arabidopsis root. The Plant Cell Online, 8, 1505
1517.
McDaniel B.K., Binder B.M. (2012) Ethylene receptor
1 (ETR1) is sufficient and has the predominant role
in mediating inhibition of ethylene responses by silver in Arabidopsis thaliana. Journal of Biological
Chemistry, 287, 2609426103.
McDonnell L., Plett J.M., Andersson-Gunner
as S.,
Kozela C., Dugardeyn J., Van Der Straeten D., Glick
B.R., Sundberg B., Regan S. (2009) Ethylene levels
are regulated by a plant encoded 1-aminocyclopropane-1-carboxylic acid deaminase. Physiologia Plantarum, 136, 94109.
Mony L., Kew J.N., Gunthorpe M.J., Paoletti P. (2009)
Allosteric modulators of NR2B-containing NMDA
receptors: molecular mechanisms and therapeutic
potential. British Journal of Pharmacology, 157,
13011317.
Muday G.K., Rahman A., Binder B.M. (2012) Auxin
and ethylene: collaborators or competitors? Trends
in Plant Science, 17, 181195.
Nahum-Levy R., Fossom L.H., Skolnick P., Benveniste
M. (1999) Putative partial agonist 1-aminocyclopropanecarboxylic acid acts concurrently as a glycinesite agonist and a glutamate-site antagonist at Nmethyl-D-aspartate receptors. Molecular Pharmacology, 56, 12071218.

Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands

Le Deunff & Lecourt

Palit S., Sharma A., Talukder G. (1994) Effects of

cobalt on plants. The Botanical Review, 60, 149181.
Piotrowski M. (2008) Primary or secondary? Versatile
nitrilases in plant metabolism. Phytochemistry, 69,
26552667.
Price M.B., Jelesko J., Okumoto S. (2012) Glutamate
receptor homologs in plants: functions and evolutionary origins. Frontiers in Plant Science, 3, 235245.
Quinn J.M., Yang S.F. (1989) Ethylene-like activity of
isocyanides. Plant Physiology, 91, 669673.
Raschke M., Boycheva S., Crevecoeur M., Nunes-Nesi
A., Witt S., Fernie A.R., Amrhein N., Fitzpatrick T.B.
(2011) Enhanced levels of vitamin B6 increase aerial
organ size and positively affect stress tolerance in
Arabidopsis. The Plant Journal, 66, 414432.
Raz V., Fluhr R. (1992) Calcium requirement for ethylene-dependent responses. The Plant Cell, 4, 1123
1130.
Romera F.J., Welch R.M., Norvell W.A., Schaefer S.C.
(1996) Iron requirement for and effects of promoters and inhibitors of ethylene action on stimulation
of Fe (III)-chelate reductase in roots of strategy I
species. BioMetals, 9, 4550.
R
uzicka K., Ljung K., Vanneste S., Podhorsk
a R.,
Beeckman T., Friml J., Benkov
a E. (2007) Ethylene
regulates root growth through effects on auxin
biosynthesis and transport-dependent auxin distribution. The Plant Cell Online, 19, 21972212.
Saftner R. (1989) Effects of organic amines on a-aminoisobutyric acid uptake into the vacuole and on
ethylene production by tomato pericarp slices. Physiologia Plantarum, 75, 485491.
Saftner R.A., Martin M.N. (1993) Transport of 1-aminocyclopropane-1-carboxylic acid into isolated
maize mesophyll vacuoles. Physiologia Plantarum,
87, 535543.
Satoh S., Esashi Y. (1983) a-Aminoisabutyric acid,
propyl gallate and cobalt ion and the mode of
inhibition of ethylene production by cotyledonary
segments of cocklebur seeds. Physiologia Plantarum, 57, 521526.
Satoh S., Yang S.F. (1989) Inactivation of 1-aminocyclopropane-1-carboxylate synthase by L-vinylglycine
as related to the mechanism-based inactivation of
the enzyme by S-adenosyl-L-methionine. Plant Physiology, 91, 10361039.
Shin K., Lee S., Song W.-Y., Lee R.-A., Lee I., Ha K.,
Koo J.-C., Park S.-K., Nam H.-G., Lee Y. (2014)
Genetic identification of ACC-RESISTANT2 reveals
involvement of LYSINE HISTIDINE TRANSPORTER1 in the uptake of 1-aminocyclopropane-1-carboxylic acid in Arabidopsis thaliana. Plant and Cell
Physiology, 56, 572582.
Sisler E.C., Pian A. (1973) Effect of ethylene and cyclic
olefins on tobacco leaves. Tobacco Science, 17, 6872.
Sisler E., Serek M. (1997) Inhibitors of ethylene
responses in plants at the receptor level: recent
developments. Physiologia Plantarum, 100, 577582.
Sisler E., Blankenship S., Guest M. (1990) Competition
of cyclooctenes and cyclooctadienes for ethylene
binding and activity in plants. Plant Growth Regulation, 9, 157164.
Soeno K., Goda H., Ishii T., Ogura T., Tachikawa T.,
Sasaki E., Yoshida S., Fujioka S., Asami T., Shimada
Y. (2010) Auxin biosynthesis inhibitors, identified
by a genomics-based approach, provide insights into
auxin biosynthesis. Plant and Cell Physiology, 51,
524536.

Ethylene inhibitors: double-edged tools

Staal M., De Cnodder T., Simon D., Vandenbussche F.,

Van Der Straeten D., Verbelen J.-P., Elzenga T., Vissenberg K. (2011) Apoplastic alkalinization is instrumental for the inhibition of cell elongation in the
Arabidopsis root by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Plant Physiology,
155, 20492055.
Staswick P.E., Tiryaki I. (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates
it to isoleucine in Arabidopsis. The Plant Cell, 16,
21172127.
Staswick P.E., Serban B., Rowe M., Tiryaki I., Maldonado M.T., Maldonado M.C., Suza W. (2005) Characterization of an Arabidopsis enzyme family that
conjugates amino acids to indole-3-acetic acid. The
Plant Cell Online, 17, 616627.
Stepanova A.N., Hoyt J.M., Hamilton A.A., Alonso
J.M. (2005) A link between ethylene and auxin
uncovered by the characterization of two root-specific ethylene-insensitive mutants in Arabidopsis. The
Plant Cell Online, 17, 22302242.
Stepanova A.N., Yun J., Likhacheva A.V., Alonso J.M.
(2007) Multilevel interactions between ethylene and
auxin in Arabidopsis roots. The Plant Cell Online,
19, 21692185.
Stepanova A.N., Robertson-Hoyt J., Yun J., Benavente
L.M., Xie D.-Y., Dolezal K., Schlereth A., J
urgens G.,
Alonso J.M. (2008) TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant
development. Cell, 133, 177191.
Strader L.C., Beisner E.R., Bartel B. (2009) Silver ions
increase auxin efflux independently of effects on
ethylene response. The Plant Cell Online, 21, 3585
3590.
Strader L.C., Chen G.L., Bartel B. (2010) Ethylene
directs auxin to control root cell expansion. The
Plant Journal, 64, 874884.
Sun P., Rane S.G., Gunasekar P.G., Borowitz J.L., Isom
G.E. (1999) Cyanide interaction with redox modulatory sites enhances NMDA receptor responses. Journal of Biochemical and Molecular Toxicology, 13,
253259.
Swarup R., Perry P., Hagenbeek D., Van Der Straeten
D., Beemster G.T., Sandberg G., Bhalerao R., Ljung
K., Bennett M.J. (2007) Ethylene upregulates auxin
biosynthesis in Arabidopsis seedlings to enhance
inhibition of root cell elongation. The Plant Cell
Online, 19, 21862196.
Tapken D., Hollmann M. (2008) Arabidopsis thaliana
glutamate receptor ion channel function demonstrated by ion pore transplantation. Journal of Molecular Biology, 383, 3648.
Tapken D., Ansch
utz U., Liu L.-H., Huelsken T., Seebohm G., Becker D., Hollmann M. (2013) A plant
homolog of animal glutamate receptors is an ion
channel gated by multiple hydrophobic amino acids.
Science Signaling, 6, ra47.
Tassoni A., van Buuren M., Franceschetti M., Fornale
S., Bagni N. (2000) Polyamine content and metabolism in Arabidopsis thaliana and effect of spermidine
on plant development. Plant Physiology and Biochemistry, 38, 383393.
Tassoni A., Watkins C.B., Davies P.J. (2006) Inhibition
of the ethylene response by 1-MCP in tomato suggests that polyamines are not involved in delaying
ripening, but may moderate the rate of ripening or
over-ripening. Journal of Experimental Botany, 57,
33133325.

Plant Biology 2015 German Botanical Society and The Royal Botanical Society of the Netherlands

Thain S.C., Vandenbussche F., Laarhoven L.J., Dowson-Day M.J., Wang Z.-Y., Tobin E.M., Harren F.J.,
Millar A.J., Van Der Straeten D. (2004) Circadian
rhythms of ethylene emission in Arabidopsis. Plant
Physiology, 136, 37513761.
Titiz O., Tambasco-Studart M., Warzych E., Apel K.,
Amrhein N., Laloi C., Fitzpatrick T.B. (2006) PDX1
is essential for vitamin B6 biosynthesis, development
and stress tolerance in Arabidopsis. The Plant Journal, 48, 933946.
Tophof S., Martinoia E., Kaiser G., Hartung W., Amrhein N. (1989) Compartmentation and transport of
1-aminocyclopropane-1-carboxylic acid and N-malonyl-1-aminocyclopropane-1-carboxylic acid in barley and wheat mesophyll cells and protoplasts.
Physiologia Plantarum, 75, 333339.
Tsang D.L., Edmond C., Harrington J.L., N
uhse T.S.
(2011) Cell wall integrity controls root elongation
via a general 1-aminocyclopropane-1-carboxylic
acid-dependent, ethylene-independent pathway.
Plant Physiology, 156, 596604.
Van de Poel B., Van Der Straeten D. (2014) 1-aminocyclopropane-1-carboxylic acid (ACC) in plants:
more than just the precursor of ethylene!. Frontiers
in Plant Science, 5, 640.
Vincill E.D., Bieck A.M., Spalding E.P. (2012) Ca2+ conduction by an amino acid-gated ion channel related
to glutamate receptors. Plant Physiology, 159, 4046.
Vincill E.D., Clarin A.E., Molenda J.N., Spalding E.P.
(2013) Interacting glutamate receptor-like proteins
in phloem regulate lateral root initiation in Arabidopsis. The Plant Cell Online, 25, 13041313.
Voesenek L.A., Harren F.J., B
ogemann G.M., Blom
C.W., Reuss J. (1990) Ethylene production and petiole growth in rumex plants induced by soil waterlogging: the application of a continuous flow system
and a laser driven intracavity photoacoustic detection system. Plant Physiology, 94, 10711077.
Voet-van-Vormizeele J., Groth G. (2008) Ethylene controls autophosphorylation of the histidine kinase
domain in ethylene receptor ETR1. Molecular Plant,
1, 380387.
Wagner S., Bernhardt A., Leuendorf J.E., Drewke C.,
Lytovchenko A., Mujahed N., Gurgui C., Frommer W.B., Leistner E., Fernie A.R., Hellmann H.
(2006) Analysis of the Arabidopsis rs4-1/pd13
mutant reveals the critical function of the PDX1
protein family in metabolism development, and
vitamin B6 biosynthesis. The Plant Cell, 18, 1722
1735.
Xu S.-L., Rahman A., Baskin T.I., Kieber J.J. (2008)
Two leucine-rich repeat receptor kinases mediate
signaling, linking cell wall biosynthesis and ACC
synthase in Arabidopsis. The Plant Cell Online, 20,
30653079.
Yang S.F., Hoffman N.E. (1984) Ethylene biosynthesis
and its regulation in higher plants. Annual Review of
Plant Physiology, 35, 155189.
Yoon G.M., Kieber J.J. (2013) 1-Aminocyclopropane1-carboxylic acid as a signalling molecule in plants.
AoB Plants, 5, plt017.
Zhao X.-C., Qu X., Mathews D.E., Schaller G.E. (2002)
Effect of ethylene pathway mutations upon expression of the ethylene receptor ETR1 from Arabidopsis. Plant Physiology, 130, 19831991.