Journal of Plant Physiology 196–197 (2016) 14–19

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Journal of Plant Physiology

journal homepage: www.elsevier.com/locate/jplph

Physiology

Co-regulation of water and K+ transport in sunflower plants during

water stress recovery
Manuel Benlloch, María Benlloch-González ∗
Departamento de Agronomía, Escuela Técnica Superior de Ingeniería Agronómica y de Montes, Universidad de Córdoba, Campus de Excelencia
Internacional Agroalimentario, ceiA3, Ctra. Madrid-Cádiz, Km. 396, E-14071 Córdoba, Spain

a r t i c l e i n f o a b s t r a c t

Article history: 16-day-old sunflower (Helianthus annuus L.) plants were subjected to deficit irrigation for 12 days. Fol-
Received 13 January 2016 lowing this period, plants were rehydrated for 2 days to study plant responses to post-stress recovery.
Received in revised form 1 March 2016 The moderate water stress treatment applied reduced growth in all plant organs and the accumulation of
Accepted 1 March 2016
K+ in the shoot. After the rehydration period, the stem recovered its growth and reached a similar length
Available online 19 March 2016
to the control, an effect which was not observed in either root or leaves. Moreover, plant rehydration
after water stress favored the accumulation of K+ in the apical zone of the stem and expanding leaves. In
Keywords:
the roots of plants under water stress, watering to field capacity, once the plants were de- topped, rapidly
Cell turgor
Deficit irrigation
favored K+ and water transport in the excised roots. This quick and short-lived response was not observed
K+ and water transport in roots of plants recovered from water stress for 2 days. These results suggest that the recovery of plant
Plant growth growth after water stress is related to coordinated water and K+ transport from the root to the apical zone
Water-stress recovery of the stem and expanding leaves. This stimulation of K+ transport in the root and its accumulation in
the cells of the growing zones of the stem must be one of the first responses induced in the plant during
water stress recovery.
© 2016 Elsevier GmbH. All rights reserved.

1. Introduction sis and as a result plant growth (Flexas et al., 2004). In addition to
this effect, stress triggers off rapid responses of growth inhibition,
Plant cells need to accumulate large amounts of K+ in the cyto- which are linked directly to the inhibition of cell elongation (Hsiao,
plasm, reaching concentrations of between 100 and 200 mM. In 1973).
many cases, this accumulation is over 400 times the average extra- According to physiological observations made over a long time,
cellular concentration (Leigh and Wyn Jones, 1984). After nitrogen, there is a close link between water and K+ flow in plants. K+ depri-
K+ is the most abundant mineral element in the cell, but unlike the vation in plants has been notice to favour water and K+ transport
former, most of the K+ is found in free ion form. As a cation, it neu- in rice (Liu et al., 2006) and sunflower (De La Guardia et al., 1985;
tralizes many of the negative electric charges generated in the cell Quintero et al., 1998) roots. More recently in this species, it has
and plays an important role in pH regulation and the activation of been observed that the removal of K+ from the root growth medium
enzymes. As a free solute, it is involved in cell osmotic regulation rapidly promotes the transport of water and K+ from the root to
(Marschner, 1995): its accumulation in the cell contributes to the the shoot, even before K+ deficiency occurs in the plant (Benlloch-
creation of the osmotic component of the water potential needed González et al., 2010). The stress caused by the confinement of the
to absorb water (Kramer, 1983), the generation of turgor and cell root system of sunflower plants in a small space also favored water
elongation (Wyn Jones et al., 1979; Hsiao and Läuchli, 1986; Shabala and K+ flow in the root (Ternes et al., 1994). In Laurus nobilis L.,
and Lew, 2002; Chen et al., 2007). The factors which cause the neg- the accumulation of K+ in the xylem of the stem has been posi-
ative effects of water stress on plant growth have been known for tively correlated with hydraulic conductivity in this organ (Nardini
a long time, and stoma closure in situations of stress is one of them et al., 2010). Short-term potassium fertilization of pot-grown laurel
(Hsiao, 1973). The lower inflow of CO2 reduces leaf photosynthe- plants cultivated under low potassium levels, favored the accu-
mulation of K+ in the xylem sap, as well as the plant’s hydraulic
conductivity and transpiration (Oddo et al., 2011). There are numer-
∗ Corresponding author. ous studies which show how the external application of ABA to
E-mail address: [email protected] (M. Benlloch-González). isolated roots quickly leads to the flow in the root of both water

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0176-1617/© 2016 Elsevier GmbH. All rights reserved.
 M. Benlloch, M. Benlloch-González / Journal of Plant Physiology 196–197 (2016) 14–19 15

(Zhang et al., 1995; Freundl et al., 1998; Hose et al., 2000; Lee et al., 2. Material and methods
2004; Sauter et al., 2002; Schraut et al., 2005) and K+ (Fournier
et al., 1987; Glinka 1980; Glinka and Abir 1983; Karmoker and 2.1. Plant material and growth conditions
Van Steveninck 1978; Quintero et al., 1998). In relation to all this
information, different studies have used CsCl, an inhibitor of K+ Sunflower seeds (Helianthus annuus L. cv. Sun-Gro 390,
channels, to study the possible co-regulation of water and K+ flow. Eurosemillas S.A., Córdoba, Spain) were surface-sterilized in 0.5%
In onion roots, this salt had an inhibitory effect on hydraulic con- (v/v) sodium hypochlorite for 1 min, and germinated in the dark for
ductivity (Tazawa et al., 2001). In rice roots, it inhibited both the 2 days at 25 ◦ C in Perlite moistened with 5 mM CaCl2 . On the sec-
accumulation of K+ and the activity of PIPs (intrinsic plasma mem- ond day, the seedlings were transferred individually to 1.5 L plastic
brane proteins) aquaporins (Liu et al., 2006). In Arabidopsis roots, it pots containing perlite and placed in a plant growth chamber with
reduced levels of the transcripts encoding the PIP and TIP (tonoplast a relative humidity between 60–80%, a day-night temperature of
intrinsic protein) water channels and the K+ transporter HAK5 (Sarh 25/22 ◦ C, a photoperiod of 14 h of light and a photosynthetic photon
et al., 2005). Along the same lines, it has been observed in rice roots flux density of 350 ␮mol m−2 s−1 (fluorescent tubes, Sylvania cool-
that K+ starvation, favored the mRNA expression levels of PIPs and white VHO). The plants were irrigated with a standard nutrient
K+ channels/transporters. In addition, in the same plant, genes of solution with the following composition: 2.5 mM Ca(NO3 )2 ; 2.5 mM
both families were down-regulated by polyethylene glycol (PEG)- KCl; 0.25 mM Ca(H2 PO4 )2 ; 1.0 mM MgSO4 ; 12.5 ␮M H3 BO3 ; 1.0 ␮M
mediated water deficit (Liu et al., 2006). All these results support MnSO4 ; 1.0 ␮M ZnSO4 ; 0.25 ␮M CuSO4 ; 0.2 ␮M (NH4 )6 Mo7 O24 and
the theory that PIP aquaporins and K+ channels/transporters are 10 ␮M Fe-ethylenediamine-di-o-hydroxy-phenylacetic acid.
functionally co-regulated. During the growing period (16 days), the plants were periodi-
Evidence showing the effect of water stress on water and K+ cally irrigated with the standard nutrient solution to dripping point.
transport in the root is, however, conflicting. In most cases, water In this way, the substrate water content was kept at field capacity.
stress inhibited hydraulic conductivity and water absorption (Aroca After this period, half of the plants were subjected to water stress
et al., 2012); however, in other studies it was observed the opposite by applying deficit irrigation. During the water stress period (12
effect (Singh and Sale, 2000; Siemens and Zwiazek, 2004). Although days), the control plants were irrigated to dripping point, while the
it is widely accepted that drought decreases the availability of K+ water stressed plants received 70 % of the amount used to water
in the soil and its absorption by roots (Shabala and Pottosin, 2014), the control ones. The water dose used to irrigate the controls was
there are many studies, mentioned above, which have described determined by measuring the difference between the water added
the positive effect of exogenous ABA on K+ and water transport in to the pot and the drainage of excess water. This procedure was
the root. Considering, as it is widely known, that drought increases repeated daily. After the water stress period, 50 % of the stressed
ABA concentration in the root (Davies and Zhang, 1991), it might plants were rehydrated for 2 days by re-watering the pots to their
seem reasonable to think that, in some circumstances, water stress field capacity. In this way, the plants were subjected to a recov-
would induce water and K+ transport in a coordinated way from ery period from water stress (Recovery). At the end of each period,
the roots to the shoot, as a strategy for avoiding stress in the shoot. the collection of the root exudates was carried out (see method in
Recently, it has been observed in different sunflower cultivars that Section 2.2).
water stress tolerance is linked to water and K+ transport in the root. At the end of the experiment, the following data was collected.
In all the cultivars studied, water stress favored water transport, Leaf area (LA) and leaf relative water content (RWC) were measured
although, in those most susceptible, K+ transport was also favored in one leaf under expansion from the third pair of leaves. LA was
(Benlloch-González et al., 2015) determined from width (W) and length (L) measurements using
Little attention has been paid to how watering after a period of the equation LA = W × L × 0.7. This coefficient was previously cal-
water stress affects water and K+ transport in the root. Some stud- culated. To determine RWC, a fresh leaf sample was first weighed,
ies has shown that aquaporins of the PIP family may be involved then rehydrated over 24 h and re-weighed according to the pro-
in plant water recovery after water stress. Antisense lines in Ara- cedure reported by Stocker (1929). The shoot (stem plus leaves)
bidopsis, with low levels of PIP1 and PIP2 aquaporins, recovered of each plant was weighed to determine fresh weight (FW). Then,
from water stress worse than controls with higher levels of these the leaves (grouped by age: growing and fully-expanded leaves)
aquaporins (Martre et al., 2002). In tobacco, water stress inhibited were frozen and stored at −20 ◦ C. The total length of the epicotyl
the expression of the aquaporins NitPIP1;1 & NitPIP2;1 as well as was also determined. In addition, two segments of approximately 5
water flow and hydraulic conductivity of the root, and watering and 10 mm long were removed from the upper (in expansion) and
after stress favored the recovery of both these hydraulic parameters lower internode of the epicotyl. Both segments (apical and basal,
and the expression of these aquaporins to normal levels (Mahdieh respectively) were individually frozen in vials until analysis.
et al., 2008).
In conclusion, there are numerous observations that suggest 2.2. Exudate collection
that roots, under different situations of stress, produce a coordi-
nated response of K+ and water flow to the shoot. However, the The exudate collection started 30 min after switching on the
information we have about the effect of water stress on these flows lights of the growth chamber. It was performed at the end of the
is both scarce and contradictory. Moreover, we do not know how water stress and rehydration-recovery period. Firstly, plants were
rehydration, after stress, affects K+ transport, and little is known de-topped 1 cm above the transition zone between the root and the
about its effect on water transport. As the plant recovers from hypocotyl, and pieces of tightly fitting latex tubing were affixed to
water stress, it probably triggers off responses to allow it to rapidly the stumps. Afterward, all the pots were irrigated with the nutri-
recover water status and growth. The quick response we know most ent solution to dripping point (Benlloch-González et al., 2010). After
about is stoma opening, which is linked to water and K+ transport a stabilization period of 6 h, the exudates from the xylem vessels
in guard cells (Fischer and Hsiao, 1968). Cell expansion in the area were collected in test tubes over an 18-h period (6th–24th h from
of stem elongation is probably another type of rapid response after the onset of exudation). During this period, the roots were kept in
stress, which involves the stimulation of water and K+ transport the same plastic pots in which the plants were grown. The collected
from the root to that area. The results obtained in this work with volume of sap was determined by measuring the difference in the
the sunflower cultivar Sun-Gro 390 support this hypothesis. weight of the test tubes, before and after the collection period. The
root-exuded sap was frozen and stored at −20 ◦ C. At the end of
16 M. Benlloch, M. Benlloch-González / Journal of Plant Physiology 196–197 (2016) 14–19

Table 1
Effect of water stress (Control vs Water stress) and rehydration after water stress
(Control vs Recovery) on shoot and root growth (g FW), leaf area (LA) (cm2 ) and
epicotyl length (cm). Plants were grown in pots with perlite substrate and watered
with a standard nutrient solution for 16 days. Then, they were subjected to water
stress by applying deficit irrigation for 12 days (Water stress). Following this period,
half of the plants were rehydrated for 2 days to recover them from water stress
(Recovery). For all treatments, values are means of 5 replicates ± standard error.

Treatment Shoot (g FW) Root (g FW) LA (cm2 ) Epicotyl length

(cm)

Control 19.7 ± 0.4 a 26.6 ± 1.4 a 71.9 ± 1.4 a 4.6 ± 0.1 a

Water stress 11.9 ± 0.8 b 15.8 ± 0.9 b 55.7 ± 1.6 b 3.4 ± 0.1 b
Significance P < 0.001 P < 0.001 P < 0.001 P < 0.001
Control 25.2 ± 0.6 a 28.4 ± 1.6 a 78.6 ± 1.8 a 5.2 ± 0.1
Recovery 15.9 ± 0.6 b 18.3 ± 1.0 b 59.5 ± 1.2 b 4.9 ± 0.2
Significance P < 0.001 P < 0.001 P < 0.001 ns

Different letters indicate significant differences at 5 %. ns = no significant differences.

the exudation, the roots were carefully recovered from the perlite
substrate and individually washed to obtain a clean root sample.
Finally, the root of each plant was weighed to determine fresh
weight (FW), before being frozen and stored at −20 ◦ C.

2.3. Other analysis

K+ was determined by atomic absorption spectrophotometry

(PerkinElmer 1100 B). It was analyzed directly in the exuded xylem
sap and the cell sap of apical and basal segments of the epicotyl. To
release the cell sap from the apical and basal segments, firstly they
were defrosted and then pressed repeatedly with a glass rod. In
the leaves (growing and fully expanded), K+ was determined after
extraction with a 10 % acetic acid solution (Benlloch et al., 1989). K+
in the xylem sap (JK ) is expressed as a flow (nmol g−1 root FW h−1 )
and was calculated from the ion concentration in the xylem sap,
xylem sap volume, fresh weight of the root, and time of exudation.
In addition, it was determined the total solute concentration of
the exuded xylem sap and the cell sap of basal and apical segments Fig. 1. Effect of water stress (Control vs Water stress) and recovery from water stress
of the epicotyl using a thermocouple psychrometer (VAPRO, Vapor (Control vs Recovery) on K+ accumulation (% DW) in growing and fully-expanded
Pressure Osmometer, 5520, WESCOR). leaves. Plants were grown in pots with perlite substrate and watered with a stan-
dard nutrient solution for 16 days. Then, they were subjected to water stress by
applying deficit irrigation for 12 days (Water stress). Following this period, half of
2.4. Experimental design the plants were rehydrated for 2 days to recover them from water stress (Recov-
ery). Measurements were made at the end of water stress and recovery period (TWS
The experiment was set up in a completely randomized design and TR respectively). For all treatments, values are means of 5 replicates ± standard
error. Different letters indicate significant differences between levels for the same
and repeated twice. In all assays, five plant replications were used
factor (P < 0.01). Means separation by Tukey’s test.
for each treatment (n = 20). The values shown are means ± SE. The
significance of the differences between the means values was deter-
mined by analyses of variance based on randomized block analysis,
using Tukey’s test and a 5 % rejection level. All statistical analyses no difference in leaf RWC between the control and recovered plants
were made using the Statistical 8.0 software package. In all analy- (90 %).
ses, residual plots were generated to identify outliers and to confirm K+ content was analyzed in growing and fully expanded leaves at
that variance was common and normally distributed. the end of the water stress treatment (TWS ) and after the recovery
period (TR ) (Fig. 1). In all cases (type of leaf and treatment), leaf
3. Results K+ content was above 1.75 % DM. While the water stress treatment
significantly reduced K+ accumulation in the fully expanded leaves,
16 day-old plants were subjected to water stress for 12 days it had no effect on the growing ones (Fig. 1). The opposite effect
(Water stress). Following this period, plants were rehydrated for 2 was observed after the recovery treatment: K+ accumulation was
days to study plant responses to post-stress recovery (Recovery). promoted only in the growing leaves (P < 0.01) and the differences
A significant reduction in shoot and root growth, LA and epicotyl in the fully expanded ones due to water stress disappeared (Fig. 1).
length was observed in water-stressed plants at the end of the The epicotyl was divided into basal and apical sections deter-
water stress period (Table 1). At this moment, the leaf RWC was mining the K+ and total osmotic solute concentration in the cell sap
91 and 85% in control and water stressed plants, respectively. The of each section (Table 2). K+ content was higher in apical than basal
rehydration-recovery period significantly promoted the growth sections at the end of both period (water stress and rehydration-
of the epicotyl stem in plants from water stress, i.e. control and recovery). K+ concentration in the basal zone was significantly
recovered plants were similar in epicotyl length after this period. reduced by water stress while no effect was observed in the api-
However, the negative effect of water stress on shoot and root cal. Recovery from water stress promoted K+ accumulation only in
growth and LA did not disappear after rehydrating the plants for 2 the apical zone of the epicotyl. Total osmotic solute concentration
days (Table 1). After this rehydration-recovery period, there were was higher in water stressed than control plants in both zones of
 M. Benlloch, M. Benlloch-González / Journal of Plant Physiology 196–197 (2016) 14–19 17

Table 2
Effect of water stress (Control vs Water stress) and rehydration after water stress
(Control vs Recovery) on K+ and total osmotic solute concentration (mM) in the
cell sap of apical and basal segments of the epicotyl. Plants were grown in pots
with perlite substrate and watered with a standard nutrient solution for 16 days.
Then, they were subjected to water stress by applying deficit irrigation for 12 days
(Water stress). Following this period, half of the plants were rehydrated for 2 days
to recover them from water stress (Recovery). For all treatments, values are means
of 5 replicates ± standard error.

K+ (mM) Osmotic solutes (mM)

Treatment Apical Basal Apical Basal

Control 103 ± 2.1 40 ± 1.7 a 339 ± 3.9 b 359 ± 5.2 b

Water stress 97 ± 1.6 28 ± 1.9 b 377 ± 4.1 a 404 ± 5.1 a
Significance ns P < 0.01 P < 0.001 P < 0.001
Control 93 ± 2.0 b 40 ± 2.2 a 367 ± 5.2 388 ± 6.9
Recovery 112 ± 1.8 a 33 ± 1.4 b 382 ± 6.8 401 ± 8.3
Significance P < 0.01 P < 0.05 ns ns

Different letters indicate significant differences at 5 %. ns = no significant differences.

Table 3
Effect of water stress (Control vs Water stress) and rehydration after water stress
(Control vs Recovery) on K+ and osmotic solutes concentration (mM) in the xylem
sap of excised roots. 16 old-day plants were subjected to water stress by apply-
ing deficit irrigation for 12 days (Water stress). Following this period, half of the
plants were rehydrated for 2 days to recover them from water stress (Recovery).
The exudates from the excised root were collected for 16 h. Numbers in brackets
indicate % of increase with respect to control. For all treatments, values are means
of 5 replicates ± standard error.

Treatment K+ (mM) Osmotic solutes (mM)

Control 6.1 ± 0.4 b 38.6 ± 1.0 b

Water stress 11.6 ± 2.2 a (90) 47.6 ± 1.7 a (23)
Significance P < 0.05 P < 0.01
Control 2.8 ± 0.2 42.4 ± 1.1
Recovery 3.5 ± 0.2 (25) 46.0 ± 1.4 (8)
Significance ns ns

Different letters indicate significant differences at 5 %. ns = no significant differences.

the epicotyl. However, this effect disappeared after rehydrating the

plants (Table 2). Fig. 2. Effect of water stress (Control vs Water stress) and recovery from water stress
Water and K+ transport (JV and JK ) in excised roots was deter- (Control vs Recovery) on water and K+ transport in excised roots (JV and JK ). Plants
mined at the end of the water stress (TWS ) and rehydration-recovery were grown in pots with perlite substrate and watered with a standard nutrient
solution for 16 days. Then, they were subjected to water stress by applying deficit
period (TR ) (Fig. 2). Immediately after each period, and once the
irrigation for 12 days (Water stress). Following this period, half of the plants were
plants were de-topped, all the pots were re-watered to field capac- rehydrated for 2 days to recover them from water stress (Recovery). At the end of
ity. In this way, the availability of water and K+ for the excised roots both periods (TWS and TR ), the plants were de-topped and then the pots with the
was the same in all the treatments. JV and JK in the excised roots excised roots were re-watered with the standard nutrient solution. In both cases,
of plant from water stress was significantly greater than in Con- the period of the exudates collection was 16 h. For all treatments, values are the
mean of 5 replicates ± standard error. Different letters indicate significant differ-
trol ones. K+ transport was three times higher in the water-stress
ences between levels for the same factor at P < 0.001 for JV and P < 0.01 for JK . Means
treatment. However, this positive effect partially disappeared in separation by Tukey’s test.
the root of the plants which underwent a 2-day recovery period:
there were no differences in JV between the control and rehydration
treatments, and JK was drastically reduced, although significant dif-
ferences were still observed (Fig. 2). Water stress also increased the tive demands, causing water deficit and loss of turgor and arresting
K+ and osmotic solute concentration in the xylem sap (Table 3). This growth (Fereres et al., 1986). Later, at night, it quickly recovers
effect was more marked for K+ concentration, with a 90 and 23% turgidity and growth (Matthews et al., 1984; Takami et al., 1982).
increase with respect to the control treatment for K+ and osmotic The osmotic water absorption by the roots, during low transpiring
concentration respectively. However, this effect disappeared after conditions, plays an important role in the recovery of the plant’s
the rehydration-recovery period (Table 3). turgor (Kramer, 1983). The ease with which it recovers growth after
a period of stress makes it an interesting plant in which to study
4. Discussion the responses immediately produced after stress.
More and more numerous observations suggest that water and
In arid areas and rain-fed agricultural ecosystems, the high tem- K+ transport from the root to the shoot is functionally coordinated
peratures lead to a high level of evapotranspiration, which often (Benlloch et al., 1983; De La Guardia et al., 1985; Ternes et al., 1994;
causes water stress in plants. These situations tend to be cycli- Quintero et al., 1998; Benlloch-González et al., 2010; Liu et al.,
cal, where states of water deficit alternate with states of turgor. 2006). However, it is not known if this phenomenon occurs per-
These cycles are often short and can even coincide with the light manently or is only induced under certain conditions. This work
and dark parts of the day. The sunflower is a species which is well has suggested that the positive effect of water stress on water and
adapted to rain-fed agricultural ecosystems in arid climates. It is K+ transport in roots during water stress recovery partly disappears
frequently subjected to high daytime temperatures and evapora- once the plant has escaped from this stress (Fig. 2).
18 M. Benlloch, M. Benlloch-González / Journal of Plant Physiology 196–197 (2016) 14–19

Some cases have been described where the aquaporins of the both flows are stimulated during stress or only once the rehydra-
PIP family may be involved in the plant’s recovery from water tion has started. Functional characterization of the physiological
stress (Martre et al., 2002; Mahdieh et al., 2008), where values of process in the plant during the period of stress is difficult, as the
water flow and hydraulic conductivity similar to those of control low availability of water and K+ in the medium prevent any possi-
plants were reached after a re-watering treatment (Mahdieh et al., ble activity of aquaporins and K+ transport in the intact plant from
2008). However, in this study, water stress and subsequent root becoming physiologically apparent. Considering that the coordi-
re-watering had a more marked effect: water flow across the root nation of water and K+ transport from the root to shoot is made
was approximately 2-fold higher in water stressed than control manifest by the action of different stresses and physiological states
plants (Fig. 2). Moreover, the results we obtained about the effect (De La Guardia et al., 1985; Ternes et al., 1994; Quintero et al., 1998;
of water stress and subsequent root re-watering on K+ transport in Liu et al., 2006; Nardini et al., 2010; Benlloch-González et al., 2010;
the root suggest that this is highly specific. The K+ concentration Oddo et al., 2011), it is logical to believe that there may be different
in the xylem sap of the roots from water stressed plants increased signaling pathways involved in this process, although in all cases,
by 90 % in comparison with the control, while the concentration the mechanism of stress perception could be the same, namely the
of total solutes only increased by 23 % (Table 3). However, after loss of turgor in the areas of cell elongation in the plant.
a 2-day rehydration-recovery period, the differences were much
smaller, which suggests that this effect is short-lived. With these
results, we cannot rule out the possibility that the stimulus to the Acknowledgment
water and K+ flow in the root occurs in the phase of water stress,
although its effect is only appreciable immediately after the stress, This study has been supported by “Proyecto de Excelencia de la
once the availability of K+ and water had increased in the medium Junta de Andalucía” grant number AGR/7152.
of the root.
It is not known what triggers these responses, but some phys- References
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