Journal of Plant Physiology 165 (2008) 1274—1279
n
of nutrient-limited plants: Its impact on
Lidiya B. Vysotskaya,
Institute of Biology, Ufa Research
Received 15 June 2007; received
Water relations of growth in favour of roots under limited nutrient supply. The ABA content was
mineral nutrients appears to be modulated by accumulation of ABA in roots. This ABA
may originate in the shoots, where its synthesis is stimulated by the loss of leaf
plant shoot growth, while root growth is often less the importance of this reaction for acclimation to
water and ion shortage is widely recognized.
Mechanisms enabling this growth allocation in
favour of roots have been assessed by Chapin
ARTICLE IN PRESS
�Corresponding author. Tel.: +7 3472355362;
fax: +7 3472356247.
(1990) and by Frensch (1997). These analyses
0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2007.08.014
E-mail address: guzel@anrb.ru (G.R. Kudoyarova).
turgor.
& 2007 Elsevier GmbH. All rights reserved.
Introduction
Shortage of mineral nutrients in soil rapidly slows
inhibited, thereby increasing the relative surface
area available for ion absorption (Chapin, 1990).
Plants also respond in this way to soil drying, and
greater in shoots and growing apical root parts of starved plants than in nutrient
sufficient plants. Accumulation of ABA in shoots of nutrient deficient plants
was linked to a decrease in leaf turgor. Increased flow of ABA in the phloem
apparently contributed to the accumulation of ABA in the apical part of the roots.
Thus, partitioning of growth between roots and shoots of wheat plants limited in
KEYWORDS
ABA;
Deficit in mineral
nutrition;
Durum wheat;
Root and shoot
growth;
Alla V. Korobova, Guzel R. Kudoyarova�
Centre, Russian Academy of Sciences, 450054 Ufa, Russian Federation
in revised form 23 August 2007; accepted 24 August 2007
Summary
We describe the involvement of abscisic acid (ABA) in the control of differential
growth of roots and shoots of nutrient limited durum wheat plants. A ten-fold
dilution of the optimal concentration of nutrient solution inhibited shoot growth,
while root growth remained unchanged, resulting in a decreased shoot/root
ratio. Addition of fluridone (inhibitor of ABA synthesis) prevented growth allocation
in favour of the roots. This suggests the involvement of ABA in the redirecting
the differential growth of roots and shoots
Abscisic acid accumulatio
in the roots
www.elsevier.de/jplph
ARTICLE IN PRESS
Abscisic acid accumulation in roots of nutrient-limited plants 1275
implicated accumulations of abscisic acid (ABA) in
bringing about the larger root/shoot ratio of
nutrient- and water-deficient plants (Chapin,
1990; Saab et al., 1990). It is therefore not
surprising that the involvement of ABA in the
control of root growth in droughted plants has
been intensively studied (e.g. LeNoble et al.,
2004). However, the likely involvement of ABA in
growth responses to deficits in major mineral
nutrients has received much less attention. Nitro-
gen and potassium deficiencies are known to
promote accumulation of ABA in root tissues of
maize (Schraut et al., 2005). However, in castor
bean, ABA was found to accumulate in shoots rather
than in roots under phosphate deficiency (Jeschke
et al., 1997). Moreover, the differences in ABA
content in nutrient-deficient plants have mainly
been discussed in relation to shoot rather than to
root growth (Dodd et al., 2002). Another feature of
this complex subject is the search for the actual
signal that induces ABA accumulation in nutrient-
deficient plants. Water shortage is well known to
induce accumulation of ABA in plants (Davies et al.,
2005). Because deficiency in mineral nutrients is
frequently accompanied by water deficit (Dodd
et al., 2002), accumulation of ABA in nutrient-
deprived plants may be due to water deficit. The
site of ABA synthesis in water-deficient plants has
been discussed in detail by Hartung et al. (2002).
ABA produced in roots of such plants was suggested
to serve as a root-derived signal to the shoot that
closes stomata and slows leaf expansion (Zhang and
Davies, 1989). Supporting evidence for such root to
shoot signalling in mineral-deficient plants revolves
around finding increased concentrations of ABA in
xylem sap of pepper and castor bean plants
following nitrate and phosphate deprivation
(Jeschke et al., 1997; Dodd et al., 2002). ABA
may also be produced in shoots and exported to
roots through the phloem (Jiang et al., 2004), its
appearance in the xylem sap resulting from
recirculation. Thus, the source of ABA in nutrient-
deprived plants remains unclear and is the topic of
the present paper, which examines the effect of
dilution of the nutrient solution on root growth,
ABA content and transport in wheat. We deter-
mined whether dilution of nutrient solution led to
accumulation of ABA in the roots and examined the
possibility that changes in leaf hydration and
osmotic potentials instigate ABA accumulation.
Experiments included treating plants with an
inhibitor of ABA synthesis (fluridone). We also
examined the effect of nutrient shortage on root
weight and branching and on indole acetic acid
(IAA) levels, since this phytohormone is important
for root growth (Reed et al., 1998).
Materials and methods
Plant material
Seedlings of durum wheat (Triticum durum Desf., cv.
Bezenchukskaya 139) were grown in containers filled with
0.1 strength (10%) Hoagland–Arnon nutrient solution
under illumination of 90Wm�2 PAR from ZN and DNAT-
400 fluorescent lamps, in a 14-h photoperiod and at
22 1C. In preliminary experiments, a 10% solution (0.5mM
KNO3, 0.5mM Ca(N03)2, 0.1mM KH2PO4, 0.1mM MgSO4,
0.5mM CaSO4) was shown to support a maximum growth
rate of wheat seedlings. Increasing the concentration of
nutrients 10-fold reduced growth in shoot dry mass by
20% over 7 d, while a 10-fold dilution to give 1% of full
strength Hoagland–Arnon solution halved growth in shoot
dry mass. Since the differences in shoot mass were
observed within 2 d, short-term changes in growth rate
and hormones were studied under limited nutrient supply
in the present experiments to reveal the primary effects.
In this work, 7-d-old plants bearing one true leaf that was
partly expanded were exposed to nutrient solution
diluted to yield 1% of full-strength Hoagland–Arnon
solution. In some experiments, fluridone was added to
the diluted nutrient solution to give a final concentration
of 5mg/L. At this stage, the shoot of plants comprised
only the first leaf. Accordingly, the terms leaf and shoot
are used interchangeably throughout the text.
Water relations and photosynthesis measurement
The leaf water potential was measured using a
Scholander-type pressure chamber. Osmotic potential of
leaves was measured by means of a freezing point
depression micro-osmometer (Camlab Limited, UK).
Turgor pressure was calculated as the difference between
water and osmotic potential. Changes in photosynthetic
CO2 assimilation and stomatal conductance were mea-
sured with a portable open system gas analyser (CIRAS-1,
PP-Systems, Hitchin, Hertfordshire, UK).
Phytohormone extraction and immunoassay
For phytohormone extraction, shoots, whole roots and
their distal growing parts (3.5mm long) were homoge-
nized in 80% ethanol and incubated overnight at +4 1C.
After filtration and vacuum evaporation of extracts to
remove all traces of ethanol, the aqueous residue was
acidified with HCl to pH 2.5 and partitioned twice with
peroxide-free diethyl ether (ratio of organic to aqueous
phases 1:3). The extracted hormones were subsequently
transferred from the organic phase into 1% sodium
hydrocarbonate (pH 7–8, ratio of organic to aqueous
phases was 3:1), re-extracted with diethyl ether,
methylated with diazomethane and immunoassayed using
polyclonal antibodies against ABA and IAA as described by
Vysotskaya et al. (2003) and Veselova et al. (2005).
Antibodies against IAA and ABA had high immunoreactiv-
ity to the corresponding hormones and low cross-
reactivity to substances structurally related to them.
Thus, in the case of immunoassay for ABA, cross-
reactivity to ABA, phaseic acid and xanthoxin was 100%,
0.1% and 0.001%, respectively, while in the case of
immunoassay for IAA, cross-reactivity to IAA, indole-3-
acetamide and indole-3-acetaldehyde was 100%, 1% and
0.15%, respectively. Reliability of immunoassay for ABA
was enabled by both the specificity of the antibodies and
purification of the phytohormones according to a
modified scheme of solvent partitioning (Veselov et al.,
1992).
Measurement of ABA flow from shoots to roots
To evaluate ABA transport from the shoot to the root,
1.5mL of 5mM Na2EDTA was applied to the base of
excised shoots to prevent plug formation in phloem sieve
root/shoot ratio similar to well-fed plants. Photo-
synthesis was inhibited by limited nutrition, while
addition of fluridone to diluted nutrient solution did
not significantly change photosynthesis and the
extent of its inhibition remained similar compared
to well-fed plants. Stomatal conductance was not
influenced by either limited supply of nutrient or
fluridone treatment, and was similar in all variants
tested. The number of lateral roots on the main
(longest seminal) root was similar in both well-fed
and starved plants. However, nutrient deficiency
decreased the number of root primordia (Figure 1).
Decreasing the availability of mineral nutrients
did not change the leaf water potentials (Table 2).
However, the osmotic pressure was lowered ap-
proximately 17% by nutrient deficit, and the turgor
pressure was higher in well-fed plants. Root
osmotic pressure was similar in well-fed and
starved wheat plants.
Dilution of nutrients did not change ABA content
in the roots as a whole, but in the apical parts
(3–4mm) levels were increased. Concentration of
ABA in the leaf was also increased two-fold by
ARTICLE IN PRESS
oto
don
tica
L.B. Vysotskaya et al.1276
elements and kept in darkness at 24 1C for 3 h (Caputo
and Barneix, 1999).
Measurement of lateral root and primordia number
Two days after dilution of the nutrient solution, lateral
roots and their primordia were counted under the
microscope in roots fixed in a mixture of ethanol and
glacial acetic acid (3:1) and stained with acetocarmine
(Vysotskaya et al., 2007).
Results
Measurement of shoot and root weight of wheat
plants 2 d after dilution of the nutrient solution
showed that a deficit in mineral nutrients inhibited
shoot growth while that of the roots remained
similar to that of well-fed plants (Table 1).
Maintaining root growth while shoot growth was
inhibited resulted in lower shoot/root ratios in
nutrient-deficient plants compared to well-fed
plants. When fluridone (an inhibitor of ABA synth-
esis) was added to the nutrient solution simulta-
neously with the dilution of nutrients, root
growth was inhibited by the nutrient deficit to
the same extent as that of the shoot, resulting in a
Table 1. Shoot and root fresh weight and their ratio, ph
2 d after dilution of nutrient solution and addition of fluri
Plant treatment Control (well-fed
plants)
Shoot weight, mg 22575
Root weight, mg 13275
Shoot/root ratio 1.7
CO2 assimilation, mmolm
�2 s�1 1171
Stomatal conductance,
mmolm�2 s�1
8674
Means of 10 replicates and their SE are presented. Means statis
(n ¼ 10).
synthesis rate and stomatal conductance of wheat plants
e
Diluted nutrient solution Diluted nutrient
solution+fluridone
20276* 19377*
13176 11174*
1.5* 1.7
671* 571*
9575 7974
lly different from control (t-test) are indicated by * (Po0.05)
Figure 1. Number of primordia (Pr) and lateral roots (LR)
per tap root of wheat plants continuously grown on 10%
(well-fed) and 2 d after their transfer to 1% (nutrient-
limited plants) Hoagland–Arnon solution (H–A).
ARTICLE IN PRESS
ave
r to
osm
ure
0.
0.
ally
(10% H–A) H–A)
Abscisic acid accumulation in roots of nutrient-limited plants 1277
ABA content in leaves 87711 178719*
ABA content in whole
roots
4574 3874
ABA content in apical
3–4mm of roots
98711 151714*
Table 2. Characteristics of water relations (MPa) in le
Hoagland–Arnon (H–A) solution and 1 d after their transfe
Nutrition Leaf water
potential
Leaf
press
Well fed (10% H–A) 0.6570.04 1.247
Nutrient limited (1% H–A) 0.6170.05 1.057
Means of 5 replicates and their SE are presented. Means statistic
Table 3. Phytohormone content in shoots and roots
(pmol g�1 fresh weight), hormone flow in xylem and
phloem (pmol plant�1 in h) of wheat plants grown
continuously on 10% Hoagland–Arnon (H–A) solution and
1 d after their transfer to 1% H–A
Well fed Limited (1%
mineral shortage (Table 3). Phloem flow of ABA to
the roots was higher in starved than in well-fed
plants, while the flow of ABA from root to shoot in
xylem sap did not change. The IAA content in the
roots was changed little by mineral deficiency.
Discussion
In our 7-d-old nutrient wheat plants, mineral
deficiency induced by diluting the nutrient solution
inhibited shoot growth over 2 d while that of the
root was kept constant. This resulted in a decrea-
sed shoot/root ratio. A similar effect has been
noted previously in tomato (Chapin, 1990) and
many other species (Wilson, 1988). Decreased shoot
growth in our nutrient-limited wheat plants may be
explained by the inhibition of photosynthesis
observed in our experiments. Reduced photosynth-
esis occurred despite no marked closing of stomata,
thus suggesting damage to the photosynthetic
apparatus itself, perhaps due to shortages of
nitrogen, potassium and other elements necessary
Xylem flow of ABA 4.570.4 5.770.6
Phloem flow of ABA 9.871 1672*
IAA content in roots 68711 5176
IAA content in apical
3–4mm of roots
251722 205730
Means of 5 replicates and their SE are presented. Means
statistically different from well-fed (t-test) are indicated by *
(Po0.05).
for photosynthesis. It is of interest that ABA
accumulation in leaves of our nutrient-limited
plants was not accompanied by a decline in
stomatal conductance, which is in apparent contra-
diction to the well-known effect of ABA inducing
the closure of stomata. However, the bulk amount
of the leaf ABA cannot be related to stomatal
conductance, which is reported to be predomi-
nantly controlled by ABA in the xylem sap (Zhang
and Davies, 1989; Hartung et al., 2002). No
increase in ABA concentration in xylem sap was
observed in our experiments, and thus the absence
of stomatal response to the limited nutrient supply
may be related to unchanged concentration of free
ABA in xylem sap of the plants.
Although dilution of nutrient solution inhibited
shoot growth, root growth remained unchanged,
resulting in a decreased shoot/root ratio. Addition
of fluridone inhibited ABA synthesis and prevented
growth allocation in favour of the roots. This
suggests the involvement of ABA in redirecting
growth in favour of roots when plants are mineral-
deficient. Fluridone may have effects in addition to
suppressing ABA production. It may therefore
influence photosynthesis. However, addition of
fluridone to diluted nutrient solution did not
change photosynthesis significantly and the extent
of its inhibition remained similar to that of well-fed
plants. When fluridone was used in experiments
with water-deficient plants as an inhibitor of ABA
synthesis by Saab et al. (1990), they too observed
that the root growth in the stressed plants
decreased, implying a positive role for ABA in root
growth. Although our measurements of the ABA in
whole roots did not reveal any changes in concen-
s and roots of wheat plants grown continuously on 10%
1% H–A
otic Turgor pressure Root osmotic
pressure
06 0.5870.03 0.4670.03
05* 0.4370.03* 0.4270.04
different from well-fed (t-test) are indicated by * (Po0.05).
tration in comparison with well-fed plants, a
marked accumulation was observed in the apical
3–4mm of the main roots. We conclude that this
ABA that accumulated in the zone of apical cell
division and expansion was sufficient to support
root growth. Saab et al. (1990) also observed an
accumulation of ABA in apical parts of roots of
droughted plants. They considered the effect being
responsible for continued elongation of roots at low
water potentials.
ARTICLE IN PRESS
L.B. Vysotskaya et al.1278
Another striking feature of the roots under
nutrient limitation was a decreased number of root
primordia formed on primary roots of our plants.
Current concepts of lateral root regulation focus on
the role of auxin (Reed et al., 1998). However, the
IAA content was unaffected by mineral shortage in
whole roots or in their apical 3–4mm. Inhibition of
lateral root growth in osmotically stressed plants
was attributed by Deak and Malamy (2005) to
‘‘interplay’’ between promotive auxin and repres-
sive ABA signalling. The initiation of root primordia
takes place in those parts of roots where we
observed accumulation of ABA in plants under
nutrient deficit. Both ABA and auxins were also
reported to be involved in the control of lateral
root growth in droughted Arabidopsis plants
(Vartanian et al., 1994). ABA stimulated the
synthesis of auxins in maize plants (Ludwig-Muller
et al., 1995). However, in our nutrient-deficient
plants the observed increase in ABA content in
root tips was not accompanied by IAA accumula-
tion, and we conclude that the unchanged level of
auxin was unable to counter the repressive effect
of ABA on lateral root formation. Thus, the
increased content of ABA in the apical 3–4mm of
nutrient-deficient roots is probably responsible for
the suppression of auxin-induced initiation of
lateral roots.
ABA accumulation in the root apices of nutrient
limited plants and its apparent involvement in
maintaining root growth raises the question of its
origin. Root-derived ABA has been reported to be
important in long-distance signalling in drought-
treated plants (Zhang and Davies, 1989; Hartung et
al., 2002; Davies et al., 2005). In the present
experiments the flow of ABA in xylem sap, the most
likely pathway for hormonal signalling from roots to
shoots, was independent of the concentration of
mineral nutrients. However, ABA is also synthesized
in shoots of plants (Creelman and Mullet, 1991) and
in our wheat plants the ABA content in leaves and
its phloem flow to the roots was higher in nutrient-
deficient plants, suggesting increased ABA synthesis
in the shoots. It has been suggested that, in
droughted plants, ABA accumulation is an outcome
of decreased shoot water potential (Zabadal,
1974). However, in the hydroponically grown
mineral-deficient wheat of the present experi-
ments, no water deficits were detected. This is in
contrast to other experiments with nutrient-defi-
cient plants grown in soil by Dodd et al. (2002).
Other studies have suggested that accumulation of
ABA in shoots may be a function of the loss of leaf
turgor (Creelman and Mullet, 1991). In the present
experiments, calculation of leaf turgor in wheat
plants revealed that it decreased in leaves of
Caputo C, Barneix AJ. The relationship between sugar
and amino acid export to the phloem in young wheat
plants. Ann Bot 1999;84:33–8.
Chapin III FS. Effects of nutrient deficiency on plant
growth: evidence for a centralised stress-response
system. In: Davies WJ, Jeffcoat B, editors. Importance
of root to shoot communication in the responses to
environmental stress. Bristol: British Society for Plant
Growth Regulation; 1990. p. 135–48.
Creelman RA, Mullet JE. Abscisic acid accumulates at
positive turgor potential in excised soybean seedling
growing zones. Plant Physiol 1991;95:1209–13.
Davies WJ, Kudoyarova G, Hartung W. Long-distance ABA
signaling and its relation to other signaling pathways
in the detection of soil drying and the mediation of the
plant’ response to drought. J Plant Growth Regul
2005;24:285–95.
Deak KI, Malamy J. Osmotic regulation of root system
architecture. Plant J 2005;43:17–28.
Dodd I, Munns R, Passioura J. Dose shoot water status
limit leaf expansion of nitrogen deprived barley. J Exp
Bot 2002;