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Water uptake by plant roots: I - Formation and propagation of a water extraction front
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in mature root systems as evidenced by 2D light transmission imaging
3
Emmanuelle Garriguesb, Claude Doussana*, Alain Pierreta
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a
INRA, Unité Climat Sol, Environnement, Bat. Sol - Domaine St Paul, Site Agroparc, 84914
Avignon cedex 9, France.
b
INRA/INAP-G, Unité Environnement et Grandes Cultures, 78850 Thiverval Grignon France
*Corresponding author: doussan@avignon.inra.fr – fax : 33 (0)4 32 72 22 12 –Phone : 33
(0)4 32 72 22 38
Key-Words : extraction front, imaging, lupin, uptake, root system, architecture, rhizotron
Text : 22 p – references: 3p – legend of figures: 2p
Figures : 9
Tables: 0
Final version: December 10, 2004
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Accepted in Plant & Soil (Springer –Verlag) – December 20, 2004 – To be published in 2005
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first submission : October 1, 2004
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1
1
Water uptake by plant roots: I - Formation and propagation of a water extraction front
2
in mature root systems as evidenced by 2D light transmission imaging
3
Emmanuelle Garriguesb, Claude Doussana*, Alain Pierreta
4
5
6
a
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Avignon cedex 9, France.
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b
INRA, Unité Climat Sol, Environnement, Bat. Sol - Domaine St Paul, Site Agroparc, 84914
INRA/INAP-G, Unité Environnement et Grandes Cultures, 78850Thiverval Grignon France
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*Corresponding author: doussan@avignon.inra.fr – fax : 04 32 72 22 12
Key-words : extraction front, imaging, lupin, uptake, root system, architecture, rhizotron
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ABSTRACT
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Soil water extraction by plant roots results from plant and soil transport processes interacting
17
at different space and time scales. At the single root scale, local soil hydraulic status and
18
plant physiology strongly control water uptake. At the whole root system level, these local,
19
spatially interacting processes, are integrated and modulated depending on the root system
20
hydraulics and plant transpiration. Most often, architectural and physiological characteristics
21
of the root system are poorly taken into account in water uptake studies. This work aims at (i)
22
studying root water extraction by mature root systems from the single root to the whole root
23
system scale and (ii) providing experimental data for the assessment of a detailed model of
24
water transport in the soil-root system presented in a companion paper (Doussan et al.,
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2005). Based on the dynamic imaging of soil water depletion around roots, we examined the
26
influence of root system architecture and soil hydraulic properties on water uptake. We
27
worked with narrow-leaf lupin plants whose root system architecture ranged from taprooted
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to fasciculate. Plants were grown in large thin containers (rhizotron) filled with a translucent
29
sand/clay mix growing medium. Water transfer in the soil, together with root water uptake,
2
1
were monitored in laboratory experiments by means of 2D light transmission imaging. This
2
technique enables the mapping of the soil water content at high spatial and temporal
3
resolutions. Throughout water uptake events, we clearly observed and quantified the
4
formation and movement of a water extraction front and of high gradients of soil water
5
content next to the roots. The data obtained also demonstrate that water uptake is never
6
restricted to a specific portion of a root and that the contribution of a specific portion of a root
7
to the overall uptake varies with time and with the position of the root within the root system.
8
Finally, we found that different root system architectures induced different water uptake
9
patterns.
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INTRODUCTION
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Water uptake by plant roots involves processes which, at the single root level, depend on
13
local soil and plant properties. At the root system level, these spatially interacting processes,
14
are integrated and modulated depending on the root system hydraulics and plant
15
transpiration. Because of the intricate nature of its underlying processes, water uptake by
16
plant roots has classically been modelled according to two main approaches: (i) the
17
“microscopic” approach (Gardner 1960), which emphasizes the role of soil for water transfer
18
towards the single root, and (ii) the “macroscopic” approach (Molz 1981) in which local
19
details are neglected and uptake is represented by a more or less empirical sink term
20
through which potential evapotranspiration is distributed over the root zone. Recently,
21
models based on the architectural description of the root system have been proposed to
22
describe the uptake of water and nutrients by plants (Clausnitzer and Hopmans 1994;
23
Doussan et al. 1999; Dunbabin et al. 2002). Such models are potentially able to give details
24
of water uptake and soil water content variations at scales ranging from the single root to that
25
of the root system. However, the way water uptake is represented differs between the
26
models. For example, Clausnitzer and Hopmans (1994) invoked a macroscopic sink term
27
located at the root apex, while Doussan et al. (1998) calculated the water flux into/along
28
roots according to the distribution of root hydraulic conductance and water potential
3
1
gradients. To validate water uptake models and the process descriptions they encapsulate,
2
experimental devices which provide information about water uptake at scales compatible with
3
the modelling (i.e. from the single root to the root system scales) are needed. Direct, real
4
time, observation of water uptake by plant roots, and water fluxes through root and stem
5
vessels is technically challenging. Hence, most research on water uptake and transport by
6
plants carried out in recent years was based on indirect methods for measuring parameters
7
which play a key role in these processes. Among others, these methods were devised to
8
measure: sap flow – with heat pulse or heat balance methods (Green and Clothier 1998);
9
plant water potential – with pressure bomb (Scholander and Hammel 1965), xylem pressure
10
probe (Balling and Zimmermann 1990), tree root suckers (Simonneau 1992), root
11
psychrometers
12
measurements (North and Nobel 1995), root pressure probe (Frensch and Steudle 1989),
13
high pressure flow meter (Tyree et al. 1993); root water uptake - with potometer (Sanderson
14
1983), dye-tracing (Varney and Canny 1993), soil water balance (Li et al. 2002). The
15
complementary value of these methods has been used to get insight into plant water
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relations, soil-plant interactions affecting water uptake as well as the possible origins of
17
heterogeneous soil water uptake by plant roots. New data gained through the use of these
18
methods also recently revived the debate over the tension-cohesion theory (Angeles et al.
19
2004).
(Vercambre
1998);
hydraulic
conductance
–
with
tension-induced
20
The common denominator of these methods is that they rely on measurements conducted
21
on excised root segments, roots or root systems separated from the soil. Due to
22
experimental constraints, results obtained by means of such methods often correspond to
23
merged information on roots the status of which (e.g. tissue age, location within the root
24
system) is not strictly similar. In contrast, imaging techniques allow the observation of live
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root systems without the need for separating any of their components from each other, or
26
from the soil in which they grow, or from the plant they are naturally attached to. The major
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experimental hindrance to imaging roots is that they are thoroughly entwined with the
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opaque, solid particles that make up the soil matrix. One alternative to bypass such a
4
1
difficulty is to render the soil transparent by using electromagnetic radiations able to
2
penetrate through the soil dense material without being completely absorbed after very short
3
travel distances through the material (Aylmore 1993). Thus, with the aid of X- or - ray
4
computed tomography (CT), breakthrough observations of the water relations at the root/soil
5
interface could be made. For example, using X-ray CT, Hainsworth and Aylmore (1989)
6
could clearly show the heterogeneous nature of water uptake along a young radish root
7
grown in a sandy soil mixture. Similarly, nuclear magnetic resonance imaging (NMRI) has
8
been successfully used to image water content variations around individual roots of pine tree
9
seedlings in sand (Macfall et al. 1990). Although technically feasible, such approaches
10
require the use of sophisticated and costly equipment. In addition, despite recent
11
technological advances, non-destructive/non-invasive observation of plant roots and their
12
environment still face a trade-off between spatial resolution, field-of-view and 3-
13
dimensionality: with the current state of the technology it is possible to have any two (Pierret
14
et al. 2003a). This is a major limitation, considering that unconstrained root systems are 3-
15
dimensional entities which include many fine roots, and which explore large volumes of soil.
16
In contrast with 3D imaging techniques such as X- or - ray and NMRI, projection imaging
17
precludes the study of fully develop three-dimensional root systems. However, provided that
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a virtually two-dimensional growing environment is an acceptable experimental condition for
19
the study of root water uptake, projection imaging permits the study of whole, fully developed
20
root systems with high spatial resolution, and does not call for the use of sophisticated
21
equipment. Up to now, this type of projection imaging has mostly been used to monitor water
22
or oil transport through soil, based on both X-ray and visible light transmission (Darnault et
23
al. 1998; Glass and Steenhuis 1989; Tidwell and Glass 1994; Tidwell and Parker 1996).
24
More recently, Pierret et al. (2003a; 2003b) presented an X-ray based projection imaging
25
setup to monitor simultaneously root growth and root water uptake. The light transmission
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projection imaging system presented in this paper has never been previously applied to the
27
study of water uptake by plant roots. We present here a simple version of this imaging
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technique which can be applied to the study of water uptake by live plants with well-
5
1
developed root systems. The proposed setup has the advantage of yielding detailed spatially
2
and temporally continuous data from the single root level to that of the root system. Such a
3
data set serves as a test for a new, detailed modelling of water uptake by plants, integrating
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root system architecture and local soil-root interactions, presented in a companion paper
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Doussan et al. (2005).
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7
MATERIALS AND METHODS
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9
Physical background of light transmission imaging
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Visible light transmission was first used quantitatively by Hoa (1981). The principle of this
11
technique is to make use of the fact that light transmission through a sandy polyphasic
12
medium (sand+air+water) increases with water content. From a physical viewpoint, the light
13
passing through the different phases of the porous medium is exponentially absorbed, but
14
also scattered and refracted at the interfaces between the phases. For the later processes,
15
the transmitted intensity of the passing light is a function of the refractive indexes of the two
16
phases and angle of incidence. When normal incidence can be assumed, Fresnel’s law gives
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the light transmission ratio ():

18
4n
(n  1) 2
(1)
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where n is the ratio of the refractive indices of two the phases. The calculated transmission
20
ratio for the sand-air (sa) and sand-water (sw) interfaces are 0.946 and 0.991 respectively
21
(Tidwell and Glass 1994). Hence, when water replaces air at sand interfaces the transmitted
22
light increases. By generalising eq. 1 to the whole porous medium, assuming that a pore is
23
either full or empty of water, Tidwell and Glass (1994) derived an equation which relates the
24
intensity Iv of transmitted light through a wet, sandy porous medium, to the water saturation
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of the medium (S):
26
 
I v  I vd   sw 
  sa 
2 Sk
(2)
6
1
where Ivd is emergent light intensity of the dry sample, k is the average number of pores
2
across the sample (k varies between 15 and 35 pores per cm depending on the sand grain
3
size). For example, a sixfold increase in transmitted light between dry and saturated states
4
might be expected from eq. 2 with k=20.
5
One important constraint of this technique is that the porous medium be translucent: for
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this reason, the thickness of experimental chamber is generally limited to less than 10 mm,
7
depending on sand grain size.
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Experimental setup
Plant material
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Narrow leaf lupin (Lupinus angustifolius L. cv Chittik) was selected because of the relative
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simplicity of its root architecture which generally consists of a taproot, abundant (but rapidly
13
decreasing with depth) primary laterals inserted more or less perpendicularly on the taproot
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and relatively few secondary laterals (Clements et al. 1993). The choice of a plant material
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with simple root architecture and minimal root overlapping was made to simplify root growth
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monitoring. Moreover, half of the work described here was a modelling exercise and, as a
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first attempt, it was desirable to keep root geometry as simple as possible.
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Seeds used in the experiments were genetically homogeneous and were selected based
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on their weight so that they all had the same growth potential. Once the rhizotrons filled with
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growing medium (see detail of experimental protocol below), pre-germinated seeds were
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placed in a central position at the top of the rhizotrons, in a hole ~5 cm deep filled with a
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mixture of perlite + peat. Artificial variations of the root system architecture were induced by
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mechanically stopping the taproot a few days after emergence with a piece of aluminium foil.
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This resulted in the emergence of 4-5 first order laterals resembling the tap root they
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originated from. The resulting architecture looked like a fasciculate root system.
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Plants were cultivated in a growth chamber in which the climatic conditions (temperature,
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relative humidity) were controlled. The photoperiod was 12 hours, the night and day
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temperatures were set at 20 and 25 °C respectively and the relative humidity was 60%.
7
1
Global radiation received by plant canopies was measured using a LiCOR 1600 porometer
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and was about 300-350 µmol PAR/m2/s (equivalent to ~150-175 W/m2). With these settings,
3
the potential evapotranspiration was 3.6 mm/day. On average plants were 50-55 days old
4
when water uptake monitoring experiments were started. Their average maximum rooting
5
depth was of the order of 80 cm and their lateral extension 46 cm. A total of 6 replicate plants
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were grown. The plants were watered daily with modified Hoagland solution, so as to
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maintain water content near field capacity. Leaf surface area was measured at the end of
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growing period with a LI-3100 area meter (Licor).
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Growing medium
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As previously mentioned, to be used in a light transmission setup, the growing medium
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must be translucent. In addition, it must satisfy constraints associated with the development
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of a plant on a very thin volume of this material. For this reason, sand only could not be used
14
because its water holding capacity would have greatly limited plant water uptake, hence plant
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development, particularly given the very small volume of the growth chamber used in this
16
work. To satisfy the translucence condition, we selected Fontainebleau quartz sand which is
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quite clear when almost pure silica and which is readily available in a wide array of grain
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sizes. Regarding the water holding capacity issue, it was decided to mix some clay with the
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sand. After testing different clay types (vermiculite, kaolinite, hectorite) / sand mixtures
20
(different grain sizes) for water holding capacity and brighness/contrast for light transmission
21
imaging, the growing medium eventually selected for the experiments was a mixture of
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98.5% (per weight) Fontainebleau sand (mean grain size: 175 µm, Prolabo) and 1.5%
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hectorite (grain size < 1 µm, Bentome Ma, Rheox). Hectorite, a nearly translucent clay
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mineral, also has a very important water retention capacity. The sand/clay mix was used as
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the growing medium in thin, transparent, rhizotrons (described below). Prior to filling up the
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rhizotrons, it was necessary to thoroughly clean the sand so that it was free of contaminants
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such as dust or grease which can greatly influence the hydrodynamic properties of the final
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mixture (Glass et al. 1989). Practically, the sand was boiled in a 0.5% detergent solution
8
1
(TFD4, Franklab S.A.) for 30-45 minutes. It was then rinsed in hot water for 15 minutes and
2
boiled another 15 minutes in tap water. Finally, the sand was washed one minute in a stream
3
of tap water and then in demineralised water. Such precautions were unnecessary with the
4
clay which is provided under purified form by the supplier. The dry, clean sand was mixed
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with the hectorite without water addition. A saturation/drying (oven at 105°C) cycle was then
6
applied to the mixture before packing it in the rhizotron.
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Rhizotron containers
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A rhizotron is generically a device which can be used to observe plant roots non-
10
destructively through transparent walls (Klepper 1992). In the specific case of light
11
transmission imaging, a rhizotron refers to an experimental container in which a growing
12
medium is introduced. The growing medium must have some properties such as a minimum
13
water holding capacity and of course be translucent. The overall thickness of the rhizotron
14
walls + growing medium must be such that light is transmitted through it at all water contents
15
and that it maximises the contrast between the dry and fully saturated ends. This can be
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easily achieved with coarse sands (> 200 µm), but as average grain size is reduced, the
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thickness of the experimental container must be reduced too so that light can still go through
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the container for the range of water contents which is desirable to explore. In the case of
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coarse sand based media, the thickness of the experimental chamber is of the order of 1 cm
20
(Tidwell and Parker 1996; Glass and Steenhuis 1989; Darnault, Throop et al. 1998). Finally,
21
rhizotron height and width must be sufficient to ensure that root development is mostly
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unconstrained (except in the 3rd dimension) during the period of time corresponding to the
23
duration of the experiments. According to these requisites, the inside dimensions of the
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rhizotrons were 1x 0.5 x 0.004 m (height x width x thickness). Rhizotron containers were
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made out of 5 mm transparent Altuglass slabs through which horizontal series of aeration
26
holes were drilled at 20 cm intervals. These holes diameter was determined so that they
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permitted air diffusion in the medium without letting grain sands through. Spacers 4 mm thick
9
1
were introduced in between the two walls to warrant a constant thickness of the growth
2
chamber. The rhizotron bottom was also drilled to permit water drainage.
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The packing process itself is of critical importance as it determines the homogeneity and
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the physical stability of the growing medium within the rhizotron during the experiment.
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Ideally, it is desirable to create an isotropic environment within the rhizotron. Practically, an
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approximation of this isotropic environment was obtained by using the protocole of Glass and
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Steenhuis (1989) in which a specifically designed funnel (which tightly fitted the shape of the
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rhizotron upper end) was used to pack the whole volume at once. It was assumed that the
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height from which the mixture falls and its encounters with the spacers distributed within the
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rhizotron resulted in a random distribution of particles. A final packing was obtained by
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bashing the top of the container 8 times with a mallet. Once packed with growing medium
12
and brought to saturation, the top of the rhizotron was covered with plastic wrapping film to
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prevent water loss by evaporation.
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Image capture
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Water uptake monitoring experiments were conducted in the same growth chamber as
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that used to grow the plants. A diagrammatic representation of the setup is given in Figure 1.
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The light source used to back-illuminate the rhizotron was a light box 1.30 m tall and 0.8 m
19
wide in which 19 neon tubes (18 W, 56 cm wide) were affixed horizontally at regular
20
intervals. A slab of white etched Altuglass (reference 27 018) positioned in front of the neon
21
tube was used as a light diffuser. A system of opaque black bellows was used to link the light
22
box to the back of the rhizotron in order to prevent light leaks which would interfere with light
23
transmission through the rhizotron itself. During the period of water uptake imaging, each
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rhizotron was placed on top of a balance (Sartorius FC) to monitor the loss of mass
25
associated with water uptake and evaporation. The monochrome CCD camera (WAT-505EX,
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Watec, 768×494 effective pixels) was attached on a photographic tripod placed ~2m away
27
from the rhizotron. The camera video signal output was subsequently digitized into 8-bit
28
format using a frame grabber board (pixel size ~1.3 mm under the configuration used to
10
1
image the rhizotrons). Since water uptake monitoring by means of light transmission strongly
2
depends upon difference imaging, it was essential to achieve the best possible image
3
registration. For this purpose, care was taken to ensure that during the period of water
4
uptake monitoring, all of the elements of the imaging chain strictly remained in the same
5
position (e.g. fixing the photographic tripod to the soil basement). It must be noted that this
6
constraint in our setup precludes the possibility of monitoring more than one rhizotron at
7
once, unless more than one imaging chain can be set up. In case one of the elements would
8
have accidentally been displaced during the experiment, registration points consisting of 4
9
cross signs were made on the rhizotron wall facing the camera lens.
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11
Water uptake experiments
12
A total of six rhizotrons were prepared. The bulk density and saturated water content of
13
the soils used were 1.650.02 g.cm-3 and 0.3790.09 cm3.cm-3 respectively. Each water
14
uptake monitoring experiment lasted for 2½ days. At the onset, the water content was set
15
close to saturation. No subsequent watering was done during the experiment. Plants were
16
submitted to a higher potential evapotranspiration (5.6 mm/day) than during the growth
17
phase by increasing the day air temperatures to 30 °C and reducing the relative humidity to
18
40%. An image was recorded approximately every 2 hours when plants were transpiring (i.e.
19
when the lights were turned on). The first image of a sequence was taken a few minutes
20
before the lights of the growth chamber were turned on. During image acquisition, it was
21
essential that no light source other than the light box be turned on in the growth chamber and
22
that camera settings be identical for all the shots (same lens aperture and shutter speed).
23
Every shot was replicated 4 times. The 4 separate images were subsequently averaged to
24
reduce the camera’s inherent thermal noise.
25
26
Image normalisation and calibration
27
Prior to applying image transforms corresponding to image calibration per se it was
28
necessary to offset parasite grey levels drifts which may arise from rapid intensity variations
11
1
of the backlight source or rapid variations of the camera’s thermal noise. This image
2
normalisation was achieved by using a test chart that includes 10 distinct grey shade bands,
3
placed next to the rhizotron within the camera field of view.
4
The relation between transmitted light intensity and water content can be established
5
empirically through a calibration approach (Darnault, Throop et al. 1998) or more
6
theoretically, based on the physics of water distribution in porous media (Tidwell and Glass
7
1994). Typically, calibration is achieved by recording images of reference samples brought to
8
known water contents. The physical and geometrical properties of these reference samples
9
must be identical to that of the medium in which experiments are run. Practically, we built a
10
special calibration chamber in which 7 separate compartments filled with the same medium
11
as that used to pack the rhizotrons were set at the following gravimetric water contents: 0, 2,
12
5, 7, 10, 15, 20 and 25%. Calibration quality was estimated by sampling 1000 pixels from the
13
image of each compartment. Figure 2 shows that the peaks corresponding to the 7
14
gravimetric water contents were clearly separated and that the grey level dispersion around
15
the grey level averages was rather low: there was no or limited overlap between peaks for all
16
water contents. To be able to derive the relation between the transmitted intensities through
17
the compartments of the calibration chamber and water contents in the rhizotrons, it must be
18
established that the medium had the same thickness and bulk density in the two containers.
19
However, in practice, it was impossible to achieve the same densities in the calibration
20
chamber, which always ended up higher than those in the rhizotrons. Since this has an
21
impact on light transmission (according to Eq. 2), this effect had to be taken into account in
22
the calibration procedure. This was done by calculating the ratio
23
normalised difference between image at near-saturation and a drier image) using Eq. 2 to
24
relate the degree of saturation S to light transmission:
I vs  I v
(which is the
I vs
25
12





  I v 

1
S
 1 
  ln 1 

s
I vs 



 2k ln  sw   
 

 sa  

1
(3)
2
3
where I v the average difference, for all the pixels, between the image of saturated and
4
drier states.
5
The calibration curve was found to correspond to two separate linear regressions, below
6
and above 8% saturation. The lower half of this calibration is less reliable as it corresponds
7
to two separate measurements only. The second part of this calibration was much more
8
satisfactory. The associated mean quadratic error of water saturation due to calibration curve
9
ranges between 0.8 and 3.9% of water saturation, equivalent to 0.003 to 0.014 cm3.cm-3
10
volumetric moisture content in our experiments. At the pixel scale, the error due to small
11
displacements of camera/rhizotron between the saturated and any later unsaturated image is
12
estimated to reach a maximum of 0.016 cm3.cm-3 volumetric water content. Consequently, at
13
the pixel scale, maximum measurement error is of the order 0.02 cm3.cm-3 volumetric water
14
content, and of the same order as found by Tidwell and Glass (1994). They give an error of
15
9% water saturation, which scales to 0.034 cm3.cm-3 water content in our experiments. At the
16
rhizotron scale, the calibrated water content compared very well (r2=0.9944) with separate
17
gravimetric water content measurements of rhizotron (Fig 3). However, we found that a bias
18
could appear giving an error of at most 0.022 cm3.cm-3 between the light transmission
19
estimated and gravimetric water content. After correcting for the bias, the residual error at the
20
rhizotron scale is 0.0025 to 0.0043 cm3.cm-3.
21
22
Results and discussion
23
24
Pattern of water content variations and uptake
13
1
Figures 4 show two examples of time series of calibrated images for two contrasted root
2
system architectures. In these images, the darker the grey levels, the drier the soil. In all
3
cases, the most important changes in water content occurred during the first day of the
4
experiments. By the end of the first day, there was already very little water left around the
5
roots located in the rhizotrons upper half. During this first day, it is conspicuous that water
6
content primarily dropped near the soil surface, although many deeper roots were present
7
and water was available at all depths. With time, water extraction occurred progressively
8
deeper. In the case of the fasciculate root system which evenly explored the volume of the
9
rhizotron, the zone of low water content spread across most of the rhizotron, after the full 2½
10
days of the experiment. Based on successive images, it could be measured that, for both the
11
taprooted and fasciculate systems, the water extraction zone expanded at an average rate of
12
3.7 cm/h during the first day, thus reaching a depth of ~40 cm by the end of this first day. It
13
must be noted though that, during the first 4 hours of the experiment, the front moved much
14
faster in the case of the taprooted root system. During the second day, the front moved at
15
average rates of only 2.2 and 0.9 cm/h for the fasciculate and taprooted root systems
16
respectively. By the end of the experiment the front was 11 cm deeper in the case of the
17
fasciculate root system. Strikingly, in the case of the taprooted root system, despite the
18
presence of numerous roots at depth more than 50 cm and the presence of the tip of the
19
taproot in the lower quarter of the rhizotron, most of the water available in the lower 50 cm of
20
the rhizotron was not extracted by the plants. The water content images were transformed
21
into soil water potential images with the retention curve of the growing medium (Doussan et
22
al., 2005). These images (data not shown) confirm that, for the taprooted root system, soil
23
water potential rapidly plummeted around the uppermost part of the root system. In contrast,
24
with the fasciculate architecture, the decline of soil water content was much less drastic and
25
extended around a much wider proportion of the root system.
26
Difference imaging is a useful means to explore the dynamics of root water uptake: it is
27
assumed here that the difference between two water content images taken at a given time
28
interval corresponds to the cumulated root water uptake over this time interval. On the basis
14
1
of such images, it is possible to locate where in the root system water uptake is occurring at
2
a given time (figure 5). Thus, it can be shown that (figure 5a), with the taprooted root system,
3
during the first 4 hours of transpiration, water uptake was concentrated in the proximal part of
4
the root system and that the most active roots were located within 15 cm of the stem base.
5
During the second part of the first day, more distal roots became actively involved in water
6
uptake: zones of water extraction tended to be increasingly localised, corresponding to a thin
7
‘crown’ of active roots that shifted ever further away from the initial uptake zone as readily
8
available water was taken up. This pattern is linked to an abrupt decrease in soil hydraulic
9
conductivity as water content falls beyond a certain value, following extraction by roots.
10
During the second and third days, the water uptake activity substantially slowed down:
11
unexpectedly, the taproot and lateral roots in the lower half of the rhizotron did not become
12
actively involved in water uptake, despite the fact that water was still available in that part of
13
the rhizotron. This is possibly because roots forming the distal part of the root system were
14
too young i.e. that their xylem vessels were not yet fully operational, thus reducing these
15
roots’ axial conductivity. The fasciculate root system (figure 5b) exhibited a similar water
16
uptake pattern although it must be noted that the zone of water uptake was more spread out
17
at all times and that the distal parts of these root systems were more actively involved in
18
water uptake than that of the taprooted systems.
19
20
Water uptake vs. transpiration (Whole plant scale)
21
Daily transpiration, as derived from calibrated images of water content, was linearly
22
correlated to plants total leaf area (r2=0.9997). According to published data (Henson and
23
Jensen 1989), transpiration rates for 50 to 65 days old blue lupin plants grown in pots could
24
range from 29 to 37.5 g/h. During the 2.5-day experiments described in this paper, observed
25
transpiration rates were on average 6-7 times less than these published values, suggesting
26
that plants grown in the 4 mm thick rhizotrons were rapidly exposed to water stress as
27
opposed to plants cultivated in larger pots. This is confirmed by the fact that during the first
28
day of the experiments, transpiration was on average 20.3 g/h (±4.2), but subsequently
15
1
plummeted to a mere 6.9 g/h (±0.7) and 3.6 g/h (±0.9) on the second and third days
2
respectively. Daily transpiration rates normalised as a function of leaf surface area (Fig 6)
3
indicate that, during the first day of the experiment, plants transpired amounts of water which
4
matched the evaporative demand (~5.6 mm/day). At this stage, the soil-root water supply to
5
the root system was non-limiting. In contrast, from the beginning of the second day and until
6
the end of the experiments, the soil-root water supply became limiting and differences
7
between root system architectures started to appear.
8
9
Influence of root system architecture on water uptake pattern (root system scale)
10
To explore whether water extraction varied depending on root system architecture, daily
11
averaged values of water uptake per unit root length (g/h/cm) were plotted against the
12
transpiration per unit leaf surface area (cm3/d/cm2). Such a comparison is based on the
13
assumption that all the roots take up water uniformly along their whole length and that all the
14
leaves transpire water at a similar rate. This assumption is of course not valid but is a useful
15
working hypothesis to test globally whether or not root architecture has an impact on water
16
uptake efficiency. This test revealed (Fig 7) that, during the first day, transpiration per unit
17
leaf surface area was virtually identical for all root architectures while water extraction per
18
unit root length was somewhat more distributed. Later on, from the second day onward, both
19
the transpiration per unit leaf surface area and the water extraction per unit root length varied
20
with root architecture. This variation can be described by a linear regression between
21
transpiration per unit leaf surface area and water extraction per unit root length. Combined
22
with information about root spatial distribution corresponding to the different root
23
architectures we experimented with (i.e. mean distance between a point in the soil and the
24
nearest root), this relation indicates that, under environmental conditions comparable to that
25
of our experiments, root systems which colonise the soil more systematically (i.e. fasciculate
26
systems) tend to take up less water when water stressed.
16
1
Similarly, when considering water uptake by the different root architectures versus soil
2
water potential, it was found that taprooted systems maintained the highest uptake rate as
3
the average soil water potential in the rhizotrons dropped from -0.1 to -0.4 MPa.
4
5
Variations in water extraction and water potential as a function of soil depth (soil profile
6
scale)
7
Most of the variation of the uptake profiles with time was apparent during the first day and
8
differences in the uptake pattern were also apparent between the taprooted and fasciculate
9
root system. With the taprooted systems, the water uptake profile consisted of a sharp peak
10
which progressively shifted to depth and the magnitude of which lessened with time (Fig 8a).
11
In contrast, for the fasciculate root system (Fig 8b), the water uptake profile was a broader
12
curve, corresponding to a larger proportion of the root system being involved in water uptake
13
at any given time. A maximum which proceeded to depth with time was still visible. This
14
uptake pattern, for all the root systems examined, denotes the formation and extension of a
15
water extraction front travelling down the root system, from the collar towards roots apical
16
ends.
17
Uptake intensity was not proportional to root length density: for example, in the fasciculate
18
system, although root length density was the same from 10 to ~50 cm, water uptake at depth
19
>25 cm was only 50% of that above at the beginning of experiment. This trend can be further
20
described by considering the maximum water extraction rate (Smax) as function of soil depth,
21
also shown in figure 8. Smax is classically calculated as:
22
S max  ETP
Lvz

Zr
0
(4)
Lvz.dz
23
where ETP is the potential evapotranspiration rate and Lvz is the root length density at
24
depth z. The root uptake rates calculated for our experiment show that the plants were able
25
to take up water at rates higher than Smax in the upper horizons (25 cm for taprooted and 35
26
cm for fasciculate), but below rates were consistently lower. This might be related to the fact
17
1
that deeper parts of the root system are younger and do not have the same water carrying
2
capacity as older tissues.
3
4
Extraction as a function of distance to roots (single root scale)
5
One advantage of light transmission imaging is that it offers an opportunity to quantify the
6
pattern of water extraction. In particular, it is possible to explore how water extraction occurs
7
as a function of distance to roots. To this purpose, the so-called Euclidean distance
8
transform (EDT) of the root image is first computed. In the transformed image, each pixel has
9
a value corresponding to its distance to the nearest root. This EDT image is then used
10
concomitantly with the calibrated image of water content to compute, for each distance lag,
11
the average water content. The result is a plot of the average water content at a given
12
distance from the nearest root and at a given time. This can be applied to all or a part of the
13
root system. Here (Fig 9), for the first day of the experiments, we explored separately 4 depth
14
increments: 0-20 cm, 20-40 cm, 40-60 cm and 60-80 cm. These plots can be interpreted as
15
the variation of water content around a virtual root which integrates the whole root system’s
16
hydraulic behaviour. It has to be noted that, on average, these plots reveal a water
17
drawdown with a the sigmoid shape similar to that obtained by Hainswoth and Aylmore
18
(1989) and Hamza and Aylmore (1992) with radish and lupin roots using X-ray tomography.
19
These authors proposed to interpret this sigmoid shape as an indication of increased soil
20
resistance to water flux as roots withdraw water from the surrounding soil. It can also be
21
seen on these plots that there existed important gradients of water content around roots (up
22
to 0.1/cm in the 0-20 cm horizon). For all the root systems, these gradients increased with
23
time, but were always larger near the surface than at depth. In the rhizotrons upper half,
24
water potential gradients were greater with taprooted than with fasciculate systems. Water
25
content near the roots could be as low as 0.05-0.1 cm3.cm-3. At such low water contents, soil
26
hydraulic conductivity dropped by about 7 orders of magnitudes compared to that at a water
27
content of 0.34 cm3.cm-3, and consequently, soil -root water transfer was very limited at some
28
distance from the roots.
18
1
It was also observed that, in rhizotrons upper half, the extent of the zone from which water
2
was extracted by individual roots was more important for taprooted than for fasciculate
3
systems (e.g 2.2 cm vs 1.8 cm in the 0-20 cm zone). Since, the soil hydraulic properties, the
4
initial water content, the evaporative demand and the root length densities at depths < 20 cm
5
(1.96 and 1.90 cm/cm3 for the taprooted and fasciculate systems respectively) were identical
6
or very similar, it can be inferred that this wider zone of influence in the case of the taprooted
7
system resulted from higher rates of root water uptake.
8
9
Discussion and conclusions
10
Compared to 3D imaging techniques such as X- or -ray CT and NMR imaging, the light
11
transmission technique presented in this paper has strong inherent limitations: the
12
experimental medium must be thin, translucent and be characterised by large enough
13
differences between its dry and wet optical properties. However, such weaknesses are
14
significantly offset by light transmission’s uniquely attractive features such as (i) very low
15
cost, (ii) setup straightforwardness, (iii) large field-of-view compatible with the root system’s
16
size of most field grown annual plants, (iv) high (virtually real time) temporal resolution
17
compatible with the study of transient water movement around plant roots, v) good spatial
18
resolution attainable with most readily available CCD cameras, and vi) dynamic range of at
19
least 8-bit (or 256 levels) depending on the type of camera used.
20
Experimentally, light transmission proved a useful tool to explore the dynamics of root
21
water uptake at the cm scale to that of the whole root system. The prominent feature
22
revealed by our experiments is the formation of a water extraction front which, with time,
23
propagated downwards and towards the distal parts of the root system. To our knowledge,
24
this is the first quantitative, time-lapse imaging of a water extraction front on a live, mature
25
root system. We could show that the formation and propagation of the extraction front
26
depend on the local soil and plant hydraulic properties. The images we obtained also
27
demonstrate that water uptake is not restricted to a specific portion of a root and that the
19
1
contribution of a specific root portion varies with time and with the position of the root in the
2
root system.
3
We also observed at a range of scales that different root architectures induced different
4
root water uptake patterns: surprisingly, under the environmental conditions of our
5
experiments, root system architectures corresponding to a more systematic spatial
6
exploration of the growing medium (and shorter mean distances between any soil point to the
7
nearest root) were found to take up less water than more taprooted systems, particularly
8
during water stress. As uptake rates per unit root length were different depending on root
9
architecture, whether the evaporative demand was met or not by the plants, it can not be
10
inferred that observed differences were simply related to the overall root system length. An
11
alternative hypothesis is that uptake rates were influenced by root spatial distribution:
12
depending on root architecture, the degree of competition between neighbour roots would
13
vary and soil water conductivity can become locally limiting (average distances to the nearest
14
root ranging from 8.2 to 11.5 cm, with values of 8.2 and 9.5 cm the taprooted and fasciculate
15
root systems, respectively). It can also be hypothesized that the observed variations in water
16
uptake rates per unit root length could have arisen from differences in root hydraulic
17
conductivities (Brar and McMichael 1990; Jones and Sutherland 1991; McCully and Canny
18
1988). Water content profiles also revealed that deeper/younger parts of the root systems did
19
not extract water as efficiently as shallower/older roots, suggesting a significant axial
20
resistance could exist in deeper roots (Pierret et al.). All these observations converge and
21
indicate that a precise description of root water uptake requires the integration of data about
22
soil and root hydraulic properties as well as root spatial distribution. In order to study all these
23
aspects of soil-root interplay on water uptake, modeling can help, in conjunction with
24
experimental work, to test these hypotheses on water transfer in the soil-root system. The
25
detail of a model combining information on architecture and hydraulic of root systems as well
26
as soil water transfer is given in a companion paper (Doussan et al., 2005) and further tested
27
with the experiments presented here.
28
20
1
2
ACKNOWLEDGEMENTS
3
This study benefited from a grant CNRS/INSU Programme National de Recherche en
4
Hydrologie n°99-PNRH-39. The authors thank V. Serra, J. Le Bot, S. Adamowitz and M.J.
5
Fabre for their assistance and technical advice.
6
7
8
21
1
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24
1
Legend of figures
2
3
4
Figure 1: Experimental setup for the light transmission imaging of water content and uptake
in a sandy soil-root system.
5
6
Figure 2: Calibration of the light transmission imaging vs water content : Histogram of the
7
intensity of transmitted light, expressed as grey levels, as function of gravimetric
8
water content of a calibration chamber. 1000 pixels are sampled on each images of
9
the different water content.
10
11
Figure 3: Comparison between gravimetric water content measurements and water content
12
estimated by the light transmission imaging by averaging the calibrated image of
13
water content over the rhizotron.
14
15
Figure 4: Images of water content variation with time in sandy rhizotrons during water uptake
16
(elapse time expressed as hours since transpiration started). The darker the image
17
the lower the water content. The rhizotrons are colonized by narrow-leaf lupin roots
18
and the plants are about 50 days old. A) Case of a tap root system – B) Case of a
19
fasciculate root system.
20
21
Figure 5: Difference images of the water content images shown figure 4 for sandy rhizotrons
22
colonized by narrow-leaf lupin roots 50 days-old. The images show the variation in
23
uptake and its location with time. The brighter the image, the greater the uptake
24
during the time period. The time is expressed as hours since transpiration started. A)
25
Case of a tap root system – B) Case of a fasciculate root system (see figure 8 too).
26
27
28
25
1
Figure 6: Mean daily transpiration of the 6 narrow-leaf lupins (~50 days old) examined in the
2
light transmission imaging experiment, during the 3 days of water uptake experiment.
3
Potential evapotranspiration is about 5.6 mm/d for the 3 days. Lupins T and F are
4
taprooted and fasciculated root systems respectively.
5
6
Figure 7: Root water uptake per unit root length as a function of daily mean transpiration of
7
the 6 narrow-leaf Lupin examined in the light transmission experiment during the 3
8
days of water uptake experiment. The first day the plants satisfy, on daily average,
9
the potential evapotranspiration. The second and third days, plant are water stressed
10
and a linear relationships emerges, related to the varying root systems architectures.
11
12
Figure 8: Profiles of water uptake rates as a function soil depth estimated from water content
13
images differences obtained with light transmission imaging (see figures 4-5). A):
14
case of a tap root system; B) case of a fasciculate root system. An extraction front
15
develops which shifts downwards with time (see also figure 5). The maximum
16
extraction rate calculated as the scaling of potential evapotranspiration by root length
17
density over depth is also presented. The plants are narrow-leaf lupin about 50 days
18
old grown in thin sandy rhizotrons.
19
20
Figure 9: Variation of water content near lupin roots. This is an average variation of water
21
content for all the roots in a 20 cm increment of soil depth. The water content are
22
estimated from light transmission images estimates of water content. The profiles at
23
different times are plotted for the taprooted and fasciculate architecture. Soil depths:
24
A): 0-20 cm; B): 20-40cm; C): 40-60 cm; D): 60-80 cm.
25
26
26