Plant Physiology Preview. Published on January 31, 2011, as DOI:10.1104/pp.110.168476
Running head: Apoplastic pH and cell elongation in the Arabidopsis root.
Corresponding author:
Prof. Dr. Kris Vissenberg
University of Antwerp
Dept. Biology, Plant Growth and Development
Groenenborgerlaan 171
B-2020 Antwerpen, Belgium
Tel: +32-3-265.34.10
Fax: +32-3-265.34.17
[email protected]
Journal Research Area: Cell Biology/Development and Hormone action
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Copyright 2011 by the American Society of Plant Biologists
1
Apoplastic alkalinisation is instrumental for the inhibition of cell
elongation in the Arabidopsis thaliana root by the ethylene precursor 1aminocyclopropane-1-carboxylic acid (ACC).
Staal Marten1,4, De Cnodder Tinne2,4, Simon Damien2, Vandenbussche Filip3, Van Der Straeten
Dominique3, Verbelen Jean-Pierre2, Elzenga Theo1, Vissenberg Kris2,5
1
Center for Ecological and Evolutionary Studies, Laboratory of Plant Physiology, University of
Groningen, P.O. Box 14, 9750 AA, Haren, The Netherlands
2
Department of Biology, Laboratory of Plant Growth and Development, University of Antwerp,
Groenenborgerlaan 171, B-2020 Antwerpen, Belgium
3
Department of Physiology, Laboratory of Functional Plant Biology, Ghent University , K.L.
Ledeganckstraat 35, B-9000 Ghent, Belgium
4
Both authors contributed equally to this manuscript
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2
Footnotes:
Financial Sources
The research was funded by the Research Foundation – Flanders (FWO; Grant G0345.02 and
G.0524.07), by the Interuniversity Attraction Poles Programme – Belgian State – Belgian Science
Policy (IUAP VI/33), the University of Antwerp and Ghent University.
5
To whom correspondence should be addressed: Prof. Dr. Kris Vissenberg, Fax: +32-3-
265.34.17, E-mail: [email protected]
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Abstract
In Arabidopsis thaliana (Col-0) roots the so called zone of cell elongation comprises two clearly
different domains: the transition zone, a post-meristematic region (approximately 200 to 450 μm
proximal of the root tip) with a low rate of elongation, and a fast elongation zone, the adjacent
proximal region (450 μm away from the root tip up to the first root hair) with a high rate of
elongation. In this study, the surface pH was measured in both zones using the microelectrode ion
flux estimation (MIFE) technique. The surface pH is highest in the apical part of the transition
zone and is lowest at the basal part of the fast elongation zone. Fast cell elongation is inhibited
within minutes by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and
concomitantly apoplastic alkalinisation occurs in the affected root zone. Fusicoccin (FC), an
activator of the plasma membrane (PM) H+-ATPase, can partially rescue this inhibition of cell
elongation, whereas the inhibitor N, N’-dicyclohexylcarbodiimide (DCCD) does not further
reduce the maximal cell length. MIFE experiments with auxin-mutants lead to the final
conclusion that control of the activity state of PM H+-ATPases is one of the mechanisms by
which ethylene, via auxin, affects the final cell length in the root.
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Introduction
Expanding and elongating plant cells are characterized by their ability to undergo wall extension
in acidic apoplastic conditions. The acid-growth theory indicates protons as the primary wall
loosening factor causing cell expansion (Rayle and Cleland, 1970; 1992). Intensive research
proved that a low apoplastic pH increases the activity of expansins in the wall, which probably
break the hydrogen bonds between the cellulose chains and the cross-linking glycans (McQueenMason et al., 1992; Cosgrove 2000). The apoplastic pH is determined by the H+-efflux through
the PM H+-ATPases and the H+-influx through H+-coupled symporters (Tanner and Caspari,
1996). Both hormonal signals such as auxin (Rayle and Cleland, 1992) and environmental cues
can affect cell growth by inducing the cell to alter its wall pH through changes in the activity of
PM H+-ATPases (Sze et al., 1999; Wu and Seliskar, 1998).
Kinematic growth analysis, with a high spatial and temporal resolution, indicated that
elongation in the Arabidopsis root is not homogenous (Beemster and Baskin, 1998). From these
results it became clear that the elongation zone can be divided into two domains with constant but
distinct growth rates (van der Weele et al., 2003). Cells close to the meristem display a very slow
elongation. Subsequently, in the second domain, there is a sudden switch to higher rates of
elongation. The first domain is called the transition zone as was suggested for the root of maize
(Baluška et al., 1996) and the adjacent zone is named the fast elongation zone. The subdivision in
a so-called ‘transition’ and ‘fast elongation zone’ was affirmed by plotting the relative elemental
growth rate along the root tip (see references in Verbelen et al., 2006). The fast elongation in the
Arabidopsis root is instantaneously inhibited by applying the plant hormone ethylene or its
precursor ACC (Le et al., 2001), and by different stresses, e.g. osmotic and salt stress (De
Cnodder et al., unpublished results).
In maize plants, the spatial profile of growth along the roots has been shown to coincide
with the spatial profile of root-surface acidification (Fan and Neumann, 2004; Peters and Felle,
1999). Peters (2004) identified the surface pH as a growth-related physiological phenomenon that
indicates the transition from a slow to fast growth. In this study, the surface pH at the root surface
was recorded along the growth zones of the Arabidopsis root during normal growth and during
ACC-induced growth arrest. The role of plasma membrane H+-ATPases in both growth
conditions was investigated as well. To our knowledge, this is the first study that correlates cell
elongation and elongation arrest with changes in the surface pH in the Arabidopsis root.
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5
Results and discussion
The surface pH and proton flux were measured along five-day-old Arabidopsis roots using the
MIFE technique (Newman, 2001; Shabala et al., 1997). In this approach the proton flux density in
or out of the root is determined by alternating the position of an H+-sensitive microelectrode
between 10 and 50 μm perpendicular to the root surface. From the pH at those two positions, and
taking into account the geometry of the root surface, the flux can then be calculated. The first
measuring point was positioned at a distance of 125 µm from the root tip to avoid interference of
the lateral root cap cells; the subsequent sampling points were 50 µm apart and the last sampling
point was at the distal border of the root hair zone.
In Figure 1A the surface pH is plotted against the position along the root. For ease of
interpretation the meristematic zone (MZ) and the two zones of elongation (transition zone, TZ
and fast elongation zone, EZ) are also indicated on the figure. The highest pH occurred at a
distance of 225 µm from the root tip. This pH maximum thus coincides with the apical limit of
the transition zone (Verbelen et al., 2006). In this zone, the surface pH decreased over 0.2 units in
a basipetal way, representing a change of 0.1 units per 100 µm distance along the root. In the fast
elongation zone the surface pH further decreased but at a slower rate of only 0.1 units over a
distance of 600 µm. The pH at a single point was stable in time (see inset in Fig. 1A). Also the
shape of the pH profile along the root was stable in time as verified by recording the pH at
different points repeatedly for a period of 3 h (results not shown). Based on these observations
and the fact that these measurements are completely uninvasive (the electrode does not even
touch the surface of the root) it is not expected that the measurements have an effect on root
functioning.
The pH profile along the root was mirrored in the H+-flux density profile (Fig; 1B). The
zone with the highest pH coincided with the zone along the root with the highest H+-influx.
Other studies have shown also a correlation between the maximal growth and the
minimal pH occurring in the growing zone of wheat (Lundegårdh, 1942) and Phleum pratense L.
roots (Monshausen et al., 1996). Simultaneous measurements of root surface pH and growth rate
along the maize root indicated the highest pH in an area just apical from the zone of explosive
growth (Felle, 1998; Peters and Felle, 1999). The latter authors found that the pH pattern along
the maize root was independent of the pH of the medium (Peters and Felle, 1999). The pH range
covered by the pH profile, however, was influenced by the pH of the medium, and increased with
an increasing medium pH. It was hypothesized by Peters (2004) that this transient pH peak
marked the transition of the root cells into an acid-growth-competent state, in which they burst
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6
into their adult size within a short period of time, a hypothesis that is clearly supported by our
results.
Thus the transition zone is the region of the Arabidopsis root in which the surface pH decreases
steeply towards a value (and the site) where fast cell elongation starts. A low apoplastic pH and
the probable activation of expansins are a prerequisite for acid-induced cell expansion to occur
(McQueen-Mason
et
al.,
1992).
Recent
results
indicate
that
also
xyloglucan
endotransglucosylase/hydrolase (XTH) proteins, capable of inducing cell wall loosening (Van
Sandt et al., 2007), can be activated at more acidic pH values as isozymes with complementary
pH activity profiles exist (Maris et al., 2009; 2010). From Vissenberg et al. (2005), Becnel et al.
(2006) and Supplemental Figure 1 (based on the Arex database; Birnbaum et al., 2003; Brady et
al., 2007) it is clear that several expansin and XTH genes are expressed in the transition and
elongation zone of the root and that they are therefore candidates to be activated by this acidic
environment.
Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC)
(Schaller and Kieber, 2002) immediately and significantly reduces root growth by the inhibition
of the fast elongation of the root cells (Le et al., 2001). This effect thus occurs in the root zone
where the surface pH has a value of about 5.4 (Fig. 1a) and slowly decreases further towards the
basis of the root. To link the ACC-induced inhibition of fast elongation with a possible effect on
wall pH, the surface pH was recorded during 2 h after ACC addition at a position between 400
and 500 µm of the root tip (i.e. the border between transition zone and fast elongation zone;
Verbelen et al., 2006). In the period from 20 min to 60 min after application the surface pH of the
root increased steeply with 0.2-0.25 pH units, and remained fairly constant afterwards (average
ΔpH after 120 min was 0.23 ± 0.02, n=3) (Fig. 2). A blank addition of medium (i.e. without
ACC) was found to have significantly less effect on the extracellular pH (average ΔpH after 120
min was 0.09 ± 0.01, n=3, student T-test p<0.005) (Fig. 2a). The effect of ACC on the root
surface pH can be fully explained by the inhibition of the H+-efflux (Fig. 2b). In the control root a
net H+-efflux is present (seen as a negative value for the H+-influx on figure 2b), while in the
ACC-treated root the efflux is completely absent. Importantly, the ACC-induced arrest of fast
elongation was not affected by keeping the root in a 10mM KCl solution in the experimental
chamber (results not shown). This rules out that the KCl solution of the measuring chamber
prevented the inhibitory effect of ACC on the root.
The ACC-induced apoplastic alkalinisation measured at the border between the transition
and the fast elongation zone thus coincides in time and space with the inhibition of the fast cell
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7
elongation. Cell wall loosening agents such as expansins that need more acidic environments now
encounter a less favourable pH environment. It is even possible that the raise in pH renders the
apoplastic environment more in favour of peroxidase activity, cross-linking specific components
of the cell wall and counter-acting the cell wall loosening enzymes. In a previous study we indeed
described cross-linking activity in the cell wall that was peroxidase-mediated and correlated with
the inhibition of cell elongation (De Cnodder et al., 2005). Furthermore, besides isozymes that
favour cell wall loosening (Van Sandt et al., 2007), it is described that some XTH proteins can
strengthen the wall (Maris et al., 2009).
In a comparable study using the MIFE technique at a single point, water deficit caused by
the addition of mannitol for 2 h to maize roots, resulted in an increase of the pH in the elongation
zone (Shabala and Newman, 1998). Moreover, in maize roots under water deficit, induced by
treatment for 48 h with polyethylene glycol (PEG 6000), the length of the zone of intense
acidification extending behind the root tip was substantially shortened (Fan and Neumann, 2004).
Our results fit with these studies. Together they clearly point to a correlation between the
apoplastic H+-concentration and the cell’s ability to elongate.
Plasma membrane H+-ATPases govern the efflux of protons across the plasma
membrane, providing the driving force for the uptake of ions and metabolites, and are thus
required for cellular growth (Palmgren, 2001). The possible link between H+-ATPase activity and
the alkalinisation of the cell wall in the ACC-growth response was investigated by modulating
this enzyme activity and checking the effect on cell elongation. Fusicoccin (FC), initially
identified as a fungal metabolite, has been shown to bind to a plasma membrane receptor complex
that includes both an H+-ATPase and a 14-3-3 protein (Alsterfjord et al., 2004; Baunsgaard et al.,
1998). FC promotes the activation of H+-ATPases, resulting in proton extrusion into the cell wall
(Olivari et al., 1998). N, N’-dicyclohexylcarbodiimide (DCCD) is a well known inhibitor of H+ATPase activity, which upon binding to the enzyme blocks the proton conductance across the
plasma membrane (Nelson and Harvey, 1999).
Measuring the length of individual trichoblast cells at the onset of root hair formation, also known
as the LEH (Length of the first Epidermal cell with visible root Hair bulge), is an easy and
suitable manner to study cell elongation in Arabidopsis roots with high temporal and spatial
resolution (Le et al., 2001). For reason of comparison, the LEH measurements were recalculated
to a ‘growth extent’ (expressed in percent) with the length of trichoblasts in control plants set as
100% (Fig. 3). Addition of ACC reduced the growth extent from 100% to 34% (Schaller and
Kieber, 2002), which was in agreement with previous findings by Le et al. (2001). However,
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8
addition of 100 µM FC to ACC-treated plants resulted in a growth extent of 86%, thus partially
rescuing cell elongation (Fig. 3), while in control plants it did not significantly affect cell
elongation (102% versus 100% respectively). This implies that the observed block in cell
elongation in ACC-treated roots was most probably in part due to the inactivation of H+-ATPase
activity. Other elongation limiting factors described by De Cnodder et al. (2005), like HRGPs
cross-linking by ROS and callose deposition in the cell wall, could be responsible for the nonreversible part of the inhibition by ACC (i.e. the missing 14% in the ACC + FC treatment).
Application of DCCD (5 µM) to control plants inhibited cell elongation bringing the growth
extent down to 53% after 3 h of treatment (Fig. 3). However, the growth extent in ACC-treated
roots was not further decreased by DCCD, indicating that ACC had already minimized the H+ATPase activity.
Taken together, the majority of plasma membrane H+-ATPases at least in the epidermis of the
elongation zone of control roots is probably in the high-activity state, whereas in ACC-treated
roots they are probably locked in their low-activity state (Sze et al., 1999). This low-activity state
of the H+-ATPases is instrumental for the measured alkalinisation of the wall pH and the
concomitant inhibition of cell elongation in ACC-treated roots.
These observations were confirmed by a set of experiments in which the pH changes
were measured at the border between the transition and the fast elongation zone in control
situations, and in situations where FC and/or ACC were added (Fig. 4). From the graphs it is clear
that FC indeed completely inhibits the alkalinisation imposed by ACC and that FC even results in
a smaller pH change than in the control situation. Furthermore, FC was able to increase the LEH
in the constitutive triple response mutant ctr1-1. In combination with the LEH measurements
described above (Fig. 3) this confirms our conclusion that the activity of H+-ATPases is necessary
in the control of cell elongation, but that additional factors indeed play a role in this process as
well.
Given the reports that ethylene controls root growth by up-regulating auxin biosynthesis
and transport-dependent auxin distribution (Ružička et al., 2007; Swarup et al., 2007), we
recorded the pH changes at the border between the transition and the fast elongation zone in roots
of wild type plants and known mutants in auxin transport (aux1-22, axr2-1 and axr3-1), both
under normal conditions and after ACC addition (Fig. 5). The results show that the auxin influx
carrier-mutant aux1-22 (Bennett et al., 1996) does not show a clear ACC-response and that the
change in pH is similar to that observed in a wild type root grown under normal conditions. This
mutant therefore seems ‘insensitive’ to the ACC application, indicating that auxin influx in cells
is necessary for the ethylene-driven inhibition of cell elongation. Untreated axr2-1 and axr3-1
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9
roots show very small pH changes that are comparable with the ones found in wild type plants.
Addition of ACC to axr2-1 roots induces a behaviour that is reminiscent of an untreated wild type
root, i.e. no alkalinisation. As described in the literature, the dominant gain of function mutant in
IAA7, axr2-1, confers auxin resistance probably by disruption of an early step in the auxin
response pathway (Wilson et al., 1990; Timpte et al., 1994). In this test we nevertheless detect a
small ACC effect, suggesting other IAAs or process might be involved. In axr3-1 ACC leads to a
hyper-alkalinisation of the root surroundings. This finding confirms the reports of a general
increase in the amplitude of auxin responses (here evoked by the ethylene treatment) in the overresponsive gain of function mutant axr3-1 (Leyser et al., 1996; Cline et al., 2001; Knox et al.,
2003). AXR3/IAA17 is a member of the Aux/IAA protein family (Rouse et al., 1998) which
generally are low abundance and labile transcription factors (Abel and Theologis, 1996). In
Figure 6 the different interactions of inhibitors and mutants of auxin-related and auxin transport
genes are depicted. The inter-relationship of auxin transport, cellular auxin levels, ethylene and
H+ efflux allows the occurrence of positive and negative feedback loops. Ethylene enhances auxin
biosynthesis and transport in the root tip (Stepanova et al., 2005; Ruzicka et al., 2007; Swarup et
al., 2007), but the regulation by downstream factors has not been described. In this context it is
interesting to note that cells and tissues that exhibit fast elongation, like pollen tubes (HoldawayClarke et al., 2003; Michard et al., 2008), root hairs
(Monshausen et al., 2007) and root
epidermal cells (Shabala and Knowles, 2002; Shabala, 2003) do show pronounced oscillations in
H+ fluxes. Although oscillations were rarely observed in the present study, the complex
interactions implied here could explain the reported oscillatory patterns in H+ fluxes in fast
growing tissues.
This set of results provides further proof of the fact that ethylene (ACC) works by
influencing the auxin content in specific cells in the treated roots. Our results strongly suggest
that this increase in cellular auxin, resulting from modified transport and/or auxin biosynthesis
(Swarup et al., 2007), in turn negatively modulates the activity of H+-ATPases which combined
with changes in the apoplast (De Cnodder et al., 2005) results in the observed cell elongation
phenotype.
Materials and Methods
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Plant material and growth conditions
Growth conditions were as described by De Cnodder et al. (2005). Five-day-old Arabidopsis Col0 seedlings and the aux1-22, axr2-1 and axr3-1 mutants were transferred to normal ½ MS media
(as a control for transfer effects; Duchefa, The Netherlands) or to media supplemented with 5 µM
1-aminocyclopropane-1-carboxylic acid (ACC; Acros Organics, Belgium) or 5 µM N, N’dicyclohexylcarbodiimide (DCCD) (dissolved in ethanol, Merck, Germany). Fusicoccin (FC)
(final concentration 100 µM, stock dissolved in ethanol; Alexis Biochemicals, USA) was applied
to the Arabidopsis seedlings in liquid ½ MS medium. Arabidopsis seedlings were placed in the
liquid medium with the cotyledons above the liquid level and the hypocotyl and the root inside
the liquid. The subsequent LEH measurements were performed as described by Le et al. (2001);
here they were recalculated to a percentual growth extent to increase the ease of interpretation.
Each experiment was repeated three times on at least 15 seedlings (n=15). To test for statistical
significance a Student’s t-test with a probability of 95 % (P=0.05) was used.
Microelectrode ion flux estimation (MIFE) technique
Measurements of the surface pH were performed as described in detail by Shabala et al. (1997).
Micropipettes (diameter 50 µm) were pulled from borosilicate glass (GC150-10, Harvard
Apparatus LTD, UK). The electrodes were silanized with tributylchlorosilane (Sigma-Aldrich,
USA) and subsequently back-filled with 15mM NaCl and 40 mm KH2PO4 and front-filled with
Hydrogen Ionophore II, Cocktail A (Sigma-Aldrich, USA). Only electrodes with a response
between 50 and 59 mV per pH unit and with a correlation coefficient between 0.999 and 1.000
(pH range 5.1-7.8) were used in experiments. A five to six-day-old Arabidopsis root from an
intact seedling was mounted on a glass capillary tube with medical adhesive B (Aromando
Medizintechnik, Germany) and placed in the experimental chamber which was filled with 1 ml of
10 mM KCl (pH 6.1). The seedling was left to recover from this manipulation for 10 minutes.
The experimental chamber was placed on an inverted microscope (Nikon TMS-F, Uvikon, The
Netherlands). The H+-microelectrode was mounted in a holder (MMT-5, Narishige, Japan) which
was attached to a micromanipulator (PCT, Luigs & Neumann, Germany) driven by a computercontrolled motor (MO61-CE08, Superior Electric, USA). The electrode was positioned manually
at a distance of 10 µm of the root surface. During the subsequent measurements, the distance
between the probe and the surface of the root was altered between 10 µm and 50 µm at a
frequency of 0.1 Hz. The chemical activity of H+ in solution at these two positions was recorded
and from these data the surface pH and the H+-flux in or out of the root was calculated. The
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11
absolute pH value could differ between different MIFE experiments, but the overall pattern of the
pH along the root stayed the same. For the sign of the H+-flux we adopted the convention by
Newman (2001), a negative sign indicates an efflux and a positive sign an influx at the root
surface. A final concentration of 5 µM ACC in the experimental chamber was attained by adding
1 µl of 5mM stock solution. As a control experiment, 1 µl of 10mM KCl was added to the
chamber. Fusicoccin was added to a final concentration 100 µM from a stock dissolved in
ethanol. In control experiments (data not shown) it was demonstrated that ethanol alone did not
affect the LEH, the proton fluxes, nor the boundary layer pH.
Supplemental Material
Supplemental figure 1
Expression patterns of members of the cell wall modifying gene families expansins and
xyloglucan endotransglucosylase/hydrolases (XTHs) in the Arabidopsis root based on the Arex
database.
Acknowledgments
The authors would like to thank Prof. Bennett (Univ. Nottingham, UK) for the generous gift of
aux1-22, axr2-1 and axr3-1 mutant seeds. F.V. is a postdoctoral fellow of the Research
Foundation Flanders (FWO).
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Figure legends
Figure 1A
Plot of the surface pH along the Arabidopsis root. The surface pH along the Arabidopsis root is
recorded using the MIFE system and sampled every 50 µm from 125 µm of the root tip off to the
root hair zone (at 1050 µm). The extracellular pH reaches a maximum at a distance of 225 µm
from the root tip and decreases towards the elongation zone. The pH values in one point are quite
stable as seen in the inset of the figure. For each single point the average was taken from a MIFE
recording lasting at least 50 seconds. MZ indicates the meristematic zone, TZ the transition zone
and EZ the fast expansion zone.
Figure 1B
Plot of the H+-flux (mean ± s.d., n=4) along the Arabidopsis root. The flux was measured with the
MIFE system as described in Figure 1a. MZ: meristematic zone, TZ: transition zone and EZ: fast
elongation zone
Figure 2A
Changes in pH (ΔpH, mean ± s.d. n=3) at a distance between 400 and 500 µm from the
Arabidopsis root tip measured during 120 min after the addition of ACC (+ ACC, final
concentration 5 µM) and after the addition of KCl as a control. The traces were recorded with a
time interval of 10 seconds between data points. For clarity symbols indicating the mean and
standard deviation bars were placed at 5 minutes intervals only. ACC addition causes an
alkalinisation in the zone of the root that marks the transition from slow to fast elongating cells.
The addition of 5 µM ACC did not significantly alter the pH of the medium in the experimental
chamber (results not shown). Before ACC addition, the sampling points at this position displayed
a steady influx and remained at a constant pH (results not shown).
Figure 2B
Effect of addition of ACC or KCl (control) on the H+-flux (mean ± s.d., n=3) measured with the
MIFE system at a distance of 400-500 μm from the root tip. Treatments were as described in
figure 2A.
Figure 3
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Modulation of the H+-ATPase activity by the activator fusicoccin (FC) and the inhibitor N, N’dicyclohexylcarbodiimide (DCCD) and its effect on the Arabidopsis root growth extent (in
percent) in control roots and in roots in which the ethylene content is increased by ACC. FC can
partially rescue the inhibition of cell elongation imposed by ACC, whereas DCCD reduces cell
lengths only in control conditions, but not in the presence of ACC. Treatments with different
lower case characters above the bars are statistically significant different at the p<0.01 level.
Figure 4
Changes in pH (ΔpH, mean ± s.d. n=3) at a distance between 400 and 500 µm from the
Arabidopsis root tip measured during 120 min in control conditions (circles) and after the
addition of ACC (filled symbols) and/or FC (triangles) and after the addition of KCl as a control
(open symbols). ACC addition causes an alkalinisation in this point along the root, whereas FC
reduces the changes in pH to a minimum, regardless of the presence of ACC
Figure 5
Changes in pH (ΔpH, mean, n=3) at a distance between 400 and 500 µm from the root tip in
Arabidopsis auxin transport mutants measured during 120 min in control conditions (open
symbols) and after the addition of ACC (filled symbols). ACC has no clear effect on the pH
changes in aux1-22 (circles), whereas the effect on pH in axr2-1 (triangles) restores the behaviour
of a wild type plant under normal conditions. Axr3-1 (squares) exhibits an hyper-alkalinisation
response after the addition of ACC. For clarity, the standard deviation bars are omitted (in general
the standard deviations were comparable with those found in figure 4).
Figure 6
Model of interactions of ethylene with different auxin response mutations (axr3-1 and axr2-1), the
auxin
transporter
mutant
(aux1-22),
the
H+-pumping
ATPase
inhibitor
N,
N’-
+
dicyclohexylcarbodiimide (DCCD) and the H -pumping ATPase activator fusicoccin (FC). In the
cartoon on the left of the root tip the disruption of auxin transport from the vascular bundle into
the root tip and from there to the cortical cells in the elongation zone by the mutation in the aux122 gene, is depicted. The box on the right shows the regulation of the proton pumping ATPase in
the cells in the elongation zone. The mutation in the aux1-22 gene prevents the accumulation of
auxin in the cell to ATPase-inhibiting levels. The gain of function mutation axr3-1 increases the
auxin responses while axr2-1 induces auxin-insensitivity. In this model ethylene is assumed to
interfere with auxin transport and biosynthesis, increasing the cellular auxin concentration to a
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18
level that is inhibitory to the H+-pumping ATPase activity. From a previous study it is known that
addition of ethylene increases ROS production and leads to HRGP cross-linking in the cell wall.
Supplemental figure 1
Expression patterns of members of the cell wall modifying gene families expansins and
xyloglucan endotransglucosylase/hydrolases (XTHs) in the Arabidopsis root based on the Arex
database. EXPA = expansin A, EXPB = expansin B, EXPLA = expansin-like A, EXPLB =
expansin-like B, XTH = xyloglucan endotransglucosylase/hydrolase
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