[Plant Signaling & Behavior 4:1, 44-46; January 2009]; 2009 Landes Bioscience

Article Addendum

Ethylene modifies architecture of root system in response to stomatal

opening and water allocation changes between root and shoot
Beauclair Patrick,1 Leblanc Antonin,1 Lemauviel-Lavenant Servane,1 Carole Deleu2 and Erwan Le Deunff1,*
1INRA;

UMR 950; Laboratoire dcophysiologie Vgtale; Agronomie & Nutritions N,C,S; Caen France;
Biotechnologies Vgtales; Rennes France

2INRA;

UMR 118; Laboratoire dAmlioration des Plantes et

Key words: ethylene, root architecture, root and shoot growth, nitrate, water relations, stomatal conductance

Ethylene plays a key role in the elongation of exploratory and

root hair systems in plants, as demonstrated by pharmacological
modulation of the activity of ethylene biosynthesis enzymes: ACC
synthase (ACS) and ACC oxidase (ACO). Thus, treatments with
high concentrations (10 M) of aminoethoxyvinylglycine (AVG,
inhibitor of ACS) and 1-aminocyclopropane carboxylic acid (ACC,
ethylene precursor, ACO activator) severely decrease the elongation
of the exploratory root system but induce opposite effects on the
root hair system: root hair length and numbers were increased in
seedlings treated with ACC, whereas they were reduced in seedlings
treated with AVG. Until now, such elongation changes of root
architecture had not been questioned in terms of nitrate uptake.
In the march issue of Plant Physiology we report that N uptake
and nitrate transporter BnNrt2.1 transcript level were markedly
reduced in ACC treated seedlings, but were increased in AVG
treated seedlings compared to the control.1 Because recent studies
have revealed that ethylene can also modulate stomatal opening as
well as root hair cell elongation, we have examined whether pharmacological modulation of ethylene biosynthesis could affect, in an
integrated manner, and at a whole-plant level, the exploratory and
root hair systems, through changes of stomatal conductance and
water allocation between the root and shoot.
The implication of ethylene in the elongation of exploratory and
root hair systems has been recognized for a long time.2,3 Recent
papers in Arabidopsis focusing on ethylene signaling during root
development, have underlined the importance of ethylene-auxin
interactions during primary root cell elongation.4-7 However while
*Correspondence to: Erwan Le Deunff; UMR INRA-UCBN 950; Laboratoire
Ecophysiologie Vgtale et Agronomie; Nutrition N, C et S; Universit de Caen;
Esplanade de la paix; 14032 Caen cdex France; Tel.: 02.31.56.53.74; Email:
erwan.ledeunff@unicaen.fr
Submitted: 10/24/08; Accepted: 10/27/08
Previously published online as a Plant Signaling & Behavior E-publication:
http://www.landesbioscience.com/journals/psb/article/7268
Addendum to: Leblanc A, Renault H, Lecourt J, Etienne P, Deleu C, Le Deunff E.
Elongation changes of exploratory and root hair systems induced by ACC and AVG
affect nitrate uptake and the BnNrt2.1 and BnNrt1.1 transporters genes expression
in Brassica napus. Plant Physiol 2008; 146:192840; PMID: 18287493; DOI:
10.1104/pp.107.109363.
44

these elegant molecular models of acropetal and basipetal movements

of auxin induced by ethylene explain root elongation, they fail to
integrate the role of the shoot and the involvement of water fluxes
within a plant via stomatal closure regulation. Indeed, recent studies
have shown that ethylene signaling is also involved in stomatal
opening.8-10 Taken together, all of these studies raise the question:
can integrated ethylene signaling from root hair to stomata at the
whole-plant level controls water fluxes and auxin movements within
the plant?11,12 In this context, our working hypothesis assumed that
ethylene could affect, in an integrated manner, the functioning of
epidermal root hair cells, as well as shoot epidermal stomatal guard
cells, and could modify water fluxes and water allocation between
the shoot and root. To test this hypothesis, we measured gaz fluxes
using a Li-COR 6400 system (Inc., Lincoln, NE) and 1 centimeter
Arabidopsis chamber (6400 s) on leaves of Brassica napus seedlings
grown on agar plates in order to establish the relationship between
changes in foliar conductance induced by different concentrations of
ACC and AVG applied to the root, and the modifications of shoot
area and root length in relation to water content.13
Changes in stomatal conductance in Brassica napus seedlings were
examined on vertical agar plates under supplies of 1 mM and 10 mM
nitrate but also under supply of 1 mM nitrate with low (1 M) or
high (10 M) concentrations of the precursor (ACC) or inhibitor
(AVG) of ethylene biosynthesis (Fig. 1A and B). The results showed
that an increase of nitrate concentration induces stomatal opening.
An identical effect is observed under the supply of 1 mM nitrate with
high ACC concentration, whereas stomatal closure is favored with
the supply of high AVG concentration compared to the control (1
mM nitrate, Fig. 1B).
This data demonstrates that at the whole-plant level, ethylene
regulates stomatal opening, as already demonstrated in detached
leaves of Vicia faba and Arabidopsis, by the use of ethylene biosynthesis inhibitors as well as ethylene mutants eto1 (ethylene over
producer).8-10 These results also confirm the effect of an increase
in nitrate concentration on stomatal opening, even if the nature
of the long distance signal responsible for this regulation is poorly
understood.11 The analogous effect of nitrate and ACC on stomatal
opening raises questions about the relationship between N metabolism and ethylene. Indeed, recent papers have reported that nitrate
and ACC treatments increase root hydraulic conductance, and that
high nitrate concentration (>10 mM) enhances both ACC oxidase

Plant Signaling & Behavior

2009; Vol. 4 Issue 1

Root and shoot architural responses to ACC treatment

activity and ACC content in roots,

suggesting ethylene has a function in
signaling in these responses.14,15
Despite an increase of stomatal
conductance, ACC treated seedlings
showed an opposing effect on shoot
area compared to nitrate treated plants
(Fig. 1C). An increasing concentration of ACC drastically reduced shoot
growth, whereas an increasing concentration of nitrate enhanced shoot
growth (Fig. 1C). The ACC growth
response has previously been explained
by the photoinhibition observed after
ACC treatment on the chlorophyll fluorescence yield (Fv/Fm) which reduces
photosystem II activity and, as a consequence, the rate of photosynthesis.16
However, the dose-response changes of
the shoot area of Brassica napus seedlings showed no effect on shoot water
content, the shoot area being correlated
with shoot water content for all the
pharmacological treatments (Fig. 1D).
Similarly, although all the treatments
reduced exploratory root length (Fig.
1E), the ACC and KNO3 treatments
induced an opposing effect on stomatal
conductance to the AVG treatment
(Fig. 1B). To interpret these paradoxical results, it is important to consider
that high concentrations of AVG can
inhibit the elongation of exploratory
and root hair systems through both
N metabolism and ethylene biosynthesis inhibition, as previously shown.1
Figure 1. Physiological and morphological changes induced after ethylene biosynthesis modulation by ACC
Indeed, a high degree of uncertainty (from 0.3 to 20 M) and AVG (from 1 to 20 M) treatments. Effects of treatments on stomatal conductance
about the targets and specificity of (B), on root length and shoot area (C and E) and on the relationships between root water mass and root
AVG treatment remains. AVG, in addi- length and shoot water mass and shoot area (D and F). Seedlings were grown on agarose gel supply with the
tion to ACC synthetase, can inhibit different concentrations treatment for 5 days after sowing. In (B) the mean se of 56 individual seedlings
many aminotransferases closely related are reported, ANOVA and Tukey test were used to compare the effect of treatments (F = 15.21, p < 0.001).
Probabilities of 0.05 or less were considered significantly different. In (D and F), the increasing concentration
to the subgroup I of aminotransferases, for each treatment is represented by the increase of symbols size. Vertical and horizontal bars indicate se
using pyridoxal 5'-phosphate (PLP) as for N = 56 seedlings when larger than the symbol.
cofactor.17,18 This lack of AVG inhibition specificity was confirmed by UPLC
analyses of root free amino acids (AA) levels in 1 mM KNO3, PLP-enzymes.1 In addition, these results demonstrate that root
10 M ACC and 10 M AVG treated seedlings. Indeed, amino hair proliferation in ACC treated plants is certainly a physiological
acids analyses showed that AVG treatment significantly increased response to the increase of stomatal opening (Fig. 1B) and the reducmajor AA such as Glutamate, Glutamine, Aspartate and Asparagine tion of the elongation of primary and lateral roots (Fig. 1A).2,3,6
compared to control and ACC treated seedlings. Despite ACC Indeed, without the possibility of increasing their shoot surface (Fig.
treatment-impaired plant growth (Fig. 1A, C and E) and C fixation, 1C) and their exploratory root system (Fig. 1E) (due to the upreguthe treatment did not significantly modify N metabolism compared lation of root auxin biosynthesis), ACC treated seedlings can only
to the control seedlings, suggesting that the ACC treatment impaired increase their root hair absorption surface to compensate for a high
transpiration rate. A similar mechanism of root surface compensation
mainly C metabolism rather than N metabolism (Table 1).16,19
In consequence, it is reasonable to assume that the reductions of for water uptake has been provided by aquaporin PIP1B antisens
stomatal conductance and root elongation observed at high AVG plants in Arabidopsis.20 In these plants, the whole exploratory root
concentrations are linked to the AVG inhibition on N metabolism system is increased five fold without alteration of the shoot biomass
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Plant Signaling & Behavior

45

Root and shoot architural responses to ACC treatment

Table 1Changes in root amino acids concentration in seedlings treated with 10 M ACC and 10 M AVG in presence
of 1 mM KNO3 during 5 days
Root amino acids

Glu
Gln
Asp
Asn
(mol. mg-1 root DW)
Control 1 mM KNO3
7.92 0.34a
3.67 0.22a
4.24 0.26b
0.85 0.05a
10 M ACC + 1 mM KNO3
6.13 0.18a
3.82 0.38a
3.07 0.15a
0.78 0.08a
10 M AVG + 1 mM KNO3
13.54 0.89b
7.26 0.78b
6.92 0.33c
1.21 0.09b

Others AA

AA tot

27.35 1.23a

44.02 1.15a

23.29 0.95a

37.09 1.53a

35.81 2.06b

64.75 3.27b

F values

47.16

15.54

59.95

9.4

18.40

43.21

p-values

p < 0.001

p < 0.001

p < 0.001

p < 0.01

p < 0.001

p < 0.001

Amino acids were extracted and analyzed according to Jubault et al., (2008).21 Values presented are mean se of four repetitions of a set of four individual seedlings. F values of Anovas were determined after assessment
of normality (Ryan-Joiner test) and homogeneity of variances (Bartlett test). Values followed by different letters are significantly different from the control conditions by means of ANOVA and Tukey tests (p < 0.05).

compared to the control. In fact, in these transformants, auxin

synthesis and its repartition along the root are certainly not affected
by the transformation event. In consequence, water flux compensation is possible via an increase in the elongation of the exploratory
root system, without modification of the root hair system as in the
roots of ACC treated plants. This hypothesis is also confirmed by
AVG treatment which increased, even at low concentrations, the
stomatal conductance as well as the elongation of the exploratory
root system (Fig. 1B and E).1,2
Taken together, all of these results suggest that ethylene affects,
in an integrated manner, the functioning of epidermal root hair
cells as well as shoot epidermal stomatal guard cells. High ACC
concentrations reduce exploratory root elongation and increase root
hair length and number to allow water flux between the root and
shoot. However, these results also raise questions about whether the
metabolisms of ethylene and nitrogen could interact in root cells
to modify exploratory and root hair systems in relation to stomatal
response and water flux within seedlings.
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