Responses of hydroponically-grown chickpea to low
phosphorus: pH changes, nutrient uptake rates, and root
morphological changes
Ghiath Alloush
To cite this version:
Ghiath Alloush. Responses of hydroponically-grown chickpea to low phosphorus: pH changes,
nutrient uptake rates, and root morphological changes. Agronomie, EDP Sciences, 2003, 23
(2), pp.123-133. .
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123
Agronomie 23 (2003) 123–133
© INRA, EDP Sciences, 2003
DOI: 10.1051/agro:2002077
Original article
Responses of hydroponically-grown chickpea to low phosphorus:
pH changes, nutrient uptake rates, and root morphological changes
Ghiath A. ALLOUSH*
Department of Soil Science and Land Reclamation, Faculty of Agriculture, Tishreen University, PO Box 1404, Lattakia, Syria
(Received 21 January 2002; revised 8 April 2002; accepted 21 May 2002)
Abstract – Plants grown in highly weathered or high alkaline soils often experience P stress, but never a P free environment. Thus, applications
of mineral P fertilizers are often required to achieve maximum yield. Responses of different plant species or cultivars were the subject of
numerous investigations in which P was withheld from the growth media. In this study, chickpea plants (Cicer arietinum L.) grown
hydroponically in complete nutrient solution for 17 days (1 mM P) were then maintained at low-P supply for 14 days (0.01 mM). Measurements
included dry matter (DM)1, production of shoots and roots, PEP carboxylase, organic acid anion (Org A–), and root morphological responses.
Dry matter of shoots and roots were reduced equally in plants subjected to Low-P supply. The uptake rates of all mineral nutrients were reduced
soon after low-P was imposed within 1–2 days for Ca2+, Mg2+, and SO42– and within 3–4 days in case of K+ and NO3–. High and low P plants
continued to alkalize the nutrient solution, but the rate of OH– released from the roots was considerably reduced in low-P plants. Cumulative
values of OH– released from the roots after low P treatment was imposed linearly correlated with the difference of anion–cation uptakes in highP (R2 = 0.9989) and in low-P plants (R2 = 0.9947). Physiological responses to low-P include an enhanced activity of PEPcase in shoots and to
a greater extent in roots, which could explain the higher concentrations of Org A– in shoots and roots of low-P plants. Root image analyses
showed an enhanced branching density in low-P plants resulting in shorter but slender roots than those in high-P plants. These responses could
probably explain the greater inflow rates and SAcR rates of micronutrient cations (Zn, Cu, Mn, and Fe) into the shoots of P-limited plants.
chickpea / phosphorus / stress / nutrient uptake / roots morphology
Résumé – Réponses du pois chiche en culture hydroponique à une faible alimentation en phosphore : changement de pH, taux
d’absorption des minéraux et modifications morphologiques des racines. Les plantes poussant sur des sols fortement altérés ou très alcalins
souffrent souvent d’un déficit de phosphore (P), mais ne se trouvent jamais dans un environnement sans P. Les applications d’engrais minéraux
phosphoriques sont donc souvent nécessaires pour obtenir un rendement maximum. Les réponses des diverses espèces ou variétés de plantes
ont fait l’objet de nombreuses recherches dans lesquelles le P a été enlevé des milieux de culture. Dans cette étude, les plants de pois chiche
(Cicer arietinum L.) cultivés hydroponiquement dans une solution nutritive complète pendant 17 jours (1 mM de P), furent ensuite maintenus
avec une faible alimentation en P pendant 14 jours (0,01 mM). Les mesures comprenaient la matière sèche des parties aériennes et des racines,
la carboxylase PEP, l’anion acide organique (Org A–), et les réponses morphologiques des racines. La matière sèche des parties aériennes et
des racines fut réduite de manière égale pour les plantes carencées. Les taux de prélèvement de tous les minéraux furent réduits rapidement
après l’imposition d’une faible alimentation en P, en 1–2 jours pour le Ca2+, le Mg2+ et le SO42– et en 3–4 jours dans le cas du K+ et du NO3–.
Les plantes, cultivées avec un fort ou un faible taux de P, ont continué d’alcaliniser la solution nutritive, mais le taux de OH– relâché par les
racines a été considérablement réduit pour les plantes carencées. Les valeurs cumulatives de OH– libérés par les racines après que les deux doses
de P aient été imposées, sont corrélées linéairement avec la différence « anion–cation » absorbés dans les plantes pour la forte (R2 = 0,9989) et
la faible (R2 = 0,9947) concentration en P. Les réponses physiologiques à une faible concentration en P comportent un accroissement d’activité
de la carboxylase PEP dans les parties aériennes et dans une plus grande mesure dans les racines, ce qui peut expliquer les fortes concentrations
de Org A– dans les parties aériennes et les racines des plantes carencées en P. Les analyses d’images de racines ont montré, de plus, un
accroissement de la densité de ramification pour les plantes carencées en P, induisant des racines plus courtes et plus fines que celles des plantes
non carencées. Ces réponses pourraient probablement expliquer les plus grands taux d’absorption et d’accumulation spécifique pour les macro
et les micronutriments (Zn, Cu, Mn et Fe) dans les parties aériennes des plantes stressées.
pois chiche / carence / phosphore / absorption racinaire / morphologie des racines
Communicated by Gérard Guyot (Avignon, France)
* Correspondence and reprints
[email protected]
1 Abbreviations: DM, dry matter; RL, root length; PEPcase, phosphoenolpyruvate carboxylase; Org A–, organic acid anion; I, inflow rate; SAcR,
specific accumulation rate.
124
G.A. Alloush
1. INTRODUCTION
Plant acquisition of nutrients of low mobility, such as
phosphorus, is often determined by their concentration in the
soil. This is especially important in highly weathered or
alkaline soils in which plants often experience P stress [24].
Annual application of P fertilizers may be required for plants
to produce reasonable growth and yield. The ability of plants
to acquire P from such soils is strongly influenced by root
morphological and physiological properties [27]. Root
morphology changes markedly in response to phosphorus
deficiency. It has been demonstrated for many plant species
(rape, bean, maize, buckwheat) that P-deficient plants have
more extensive root systems, which are characterized by more
rapid development, higher root/shoot ratios, and finer and
longer roots, as well as more root hairs [3, 7, 9, 31]. These
changes in the root structure under P-stress allowed more
efficient acquisition of P, which was attributed to a greater
ability to explore the soil, and hence improve P recovery [24].
Similarly, there are a number of functional changes in the
roots that result from P deficiency, which also enhance the
uptake of soil P.
P-fixation in the soil [24]. Therefore, depending on soil
management, soil type, and environmental factors, plants may
experience low-P availability in the soil later in the growing
season rather than absolutely P-free soil solution. In this study,
non-nodulated chickpea plants were grown initially with high
rate of P supply (1 mM P) to establish relatively sizable plants,
and then subjected to low-P supply (0.01 mM P), rather than
complete P-starvation [12, 14, 20, 25, 27]. The Rhizobium
symbiosis with chickpea roots was intentionally avoided in
this study as may alter root physiological and morphological
responses [24]. The overall objectives were to investigate:
(i) the extent of growth reduction and mineral nutrient uptakes
due to low-P supply; (ii) the speed response to low-P stress in
acidifying the growth media; (iii) the occurrence of root morphological changes; and (iv) chickpea physiological responses
with special reference to activity of PEP carboxylase enzyme
and subsequent accumulation of organic acids in plant tissues.
2. MATERIALS AND METHODS
2.1. Plant cultivation
Evidence for acidification of the substrate has been
observed for many plant species in response to P deficiency.
Moorby et al. [25] demonstrated using rape plants (Brassica
napus L.), that acidification was the results of net H+ excretion
from the roots consequent to the shift in cation/anion uptake
ratio, although the uptake of anion remained slightly higher
than cation. Such acidification has also been observed (though
NO3− was the main source of nitrogen supply) for chickpea
plants (Cicer arietinum L.) grown in nutrient solution [20],
and for tomato plants [12]. The supply of NO3−-N normally
increases anion over cation uptake and nitrate may represent
approximately 80% of the total anion uptake by tomato
plants [18]. In all cases, the observed acidification of substrate
by different plant species coincided with accumulation and/or
release of organic acid anions [12, 20, 25, 27]. Hoffland
et al. [15] concluded that acidification of the rhizosphere
resulted from the release of organic acids, which was matched
by an increase in activity of PEP carboxylase enzyme in rape
plants. Organic acids also play an important role in plant
physiology and maintain intercellular pH and electrochemical
charge [19]. An enhanced activity of the enzyme PEP
carboxylase was reported in several plant species subjected to
P stress such as rape, tomato, and lupin [15, 16, 27]. Some
other plant species, such as white clover and buckwheat, were
shown to accumulate sufficient P during periods of high
supply to maintain growth through times without adequate
availability of P [3, 6]. However, conclusions regarding the
importance of each of these P-deficiency response
mechanisms have been contradictory.
The complete nutrient solution (H-P) consisted of
Ca(NO3)2, 1.5 mM; KH2PO4, 1.0 mM; and MgSO4, 0.5 mM.
The L-P nutrient solution consisted of Ca(NO3)2, 1.5 mM;
KH2PO4, 0.01 mM; MgSO4, 0.5 mM and K2SO4, 0.495 mM
to compensate for K. The concentration of sulfate, an anion
known to have little effect on the uptake of other
macronutrients was thus varied in the two P nutrient solutions.
To both nutrient solutions (Low and High P), micronutrients
were supplied according to the Long Ashton formula [13].
The extent of these response mechanisms is probably
important when plants are subjected to P-free growth substrates [12, 20, 25, 27]. It is most unlikely for plants growing
in soils to experience absolute P-free environment and therefore, it is a matter of speculation on the extent appearance of
these functional responses if plants do not experience absolute
P starvation. The availability of P in the soil is mainly determined by the initial plant uptake and simultaneously occurring
Every 24 hours at the beginning of each light cycle, the
circulation pumps were stopped for 5 min to allow the used
nutrient solutions to drain into the reservoir tanks, after which
they were replaced with 10 L fresh low or high P solutions
(pH = 6.0) according to treatments. At the beginning and end
of each cycle, 50 ml samples from each tank were taken for
mineral nutrient concentration analyses. The volumes of used
nutrient solutions, and pH, were measured. Using 1-L of the
Chickpea (Cicer arietinum L. cv. UC 27) seeds were
surface sterilized (4 min in 10% sodium hypochlorite, with
5 drops of teepol followed by 5 rinses in deionized water) and
germinated in a moist peat-perlite mixture. Eighty seedlings
(0.74 ± 0.04 fresh wt.basis) were selected and transferred to
plastic troughs, along which nutrient solution was circulated
from reservoir tanks. Each trough supported 10 seedlings and
each reservoir tank held 10 L of nutrient solution, which was
fed into four troughs at a flow rate of 2 L·min−1. After a 7-day
pre-culturing period, during which half-strength, complete
nutrient solution was circulated, 2 seedlings from each trough
were harvested to begin the experiment (t0). Plants in all
troughs (8 in each trough) were subsequently supplied with
full-strength, complete nutrient solutions for 13 days. One of
two treatment regimes was then imposed: (1) in four troughs,
24 plants continued to receive the complete nutrient solution
containing 1 mM P (H-P, high-P treatment), and (2) 24 plants
were P-limited by reducing the P concentration in the nutrient
solution to 0.01 mM P (L-P, low-P treatment).
Responses of hydroponically-grown chickpea to low P
used nutrient solutions, the pH was titrated back to the starting
pH (6.0) using 0.5 mM HCl. The quantities of acid necessary
to correct the pH of the nutrient solutions at each cycle were
recorded and results expressed as rate (meq·plant−1 ·day−1)
and cumulative OH− released (meq·plant−1) from the roots.
125
the PEP was followed in a spectrophotometer at 340 nm.
Protein concentrations were determined by the methods of
Bradford [5].
2.3. Root digital image analyses
2.2. Procedure at harvest and measurements
Four plants from each treatment, selected randomly, were
harvested at 0, 9, 13, 17, 21, and 27 days. At each harvest,
roots were cut from shoots and fresh weights were recorded. A
known fresh weight from shoots and roots was taken for PEP
carboxylase activity, and from roots for total root length (RL),
determined using the RL scanner (Comair Model TM0001,
Melbourne, Australia). The remaining fresh matter of shoots
and roots were oven-dried at 70 °C for at least 48 h, and total
dry matter (DM) of shoots and roots was then calculated.
Shoots and roots were ground to pass a 0.5-mm screen and
kept in sealed plastic bags for total and soluble mineral ion
concentration determinations. Between 50−100 mg of plant
samples were weighed into teflon containers, after which
1.0 mL of 15.8 M HNO3 was added. The samples were then
digested using the microwave procedure [16]. Digested
samples were brought to a final volume of 10.0 mL, filtered,
and analysed for total mineral elements (P, S, K, Ca, Mg, and
Na) using Inductive Couple Plasma (ICP, Jobin Yvon Model
JY 46P ICP, Longjumeau, France). Total nitrogen contents in
shoot and root tissues were determined by using a Carlo Erba
analyzer (EA 1108 CHNSO, Milan, Italy).
Soluble NO3− and Cl− were determined from hot water
extracts using an ion chromatograph [2] with suppressed
conductivity (Dionex DX 500 ion chromatography, AS 14
4 mm anion column and CS 14 4mm cation column).
Alkalinity of the tissues [34] was determined by ashing
subsamples (250–500 mg) in porcelain crucibles at 500 °C for
6 h. Samples were then allowed to cool and the ash dissolved
in 10 mL of 1 M standardized HCl, followed by back titration
to pH 7.0 using 1 M NaOH solution. The calculated difference
in volume between the two standard solutions (acid–alkaline)
represents the alkalinity of the ash and is taken as a good
estimator of the organic acid content of the plant tissues.
Xylem sap was collected from additional three plants from
L-P and H-P treatments at day 27. Collection of sap was made
over a period of 5 minutes from each plant using a pressure
bomb (Soil Moisture Equipment Crop, USA) at 2–4 psi. Sap
samples were analyzed for mineral nutrient concentrations
using ICP for (P, S, K, Ca, and Mg) and using Dionex
chromatography for anions (NO3−, Cl–, SO4−2, and H2PO4−).
The extraction and assay of PEPcase was performed
according to Marques et al. [23], and as modified by
Schweizer and Erismann [32]. Desalted extracts of shoots and
roots were incubated with PEP, malate dehydrogenase
[EC1.1.1.37] and NADH, and the disappearance of NADH
during the conversion to malate of oxalacetate formed from
2
Mention of trademark does not imply endorsement by USDA over
comparable products.
Two plants were taken from Low and High-P treatments at
each harvest after P-stress was imposed (17, 21, and 27 days).
Triplicates of basal roots were teased from the main root axes
of each chickpea plant, arranged using a small paint brush in a
glass tray partially filled with distilled water and placed on top
of a light box (Aristo model DA-10, Rosyln NY, USA). A
digital image of the entire root was recorded using a Dage 72S
CCD (charge coupled device) camera (Dage-MTI Inc.,
Michigan City, IN, USA), fitted with a Minolta 50/1.7 lens.
Image capture was controlled by a Power Macintosh 7200/
120PC compatible computer equipped with an AG-5 frame
grabber board (Scion Inc., Frederick, MD, USA) and NIHImage (version 1.60 adapted by Scion, Inc. from public
domain software). In order to investigate the effects of P-stress
on lateral root branching, density, length, branch root
characteristics were recorded from a 12–15 cm region of the
root where branches first appeared from the apical meristem.
2.4. Calculations and statistical analyses
Uptake of macronutrient elements over a 24-h period was
calculated from differences between concentrations at the start
and end of each 24-h depletion cycle, multiplied by the
volume of the solution in each trough system and reservoir
tank. The results of these calculations give the uptake rates
of nutrients (mg·plant−1 ·day−1), when divided by number of
plants remaining in each trough system and tank. The sum of
uptake rates yield the cumulative uptake of nutrient elements
(mg·plant−1) and are average values and statistical analyses
could not be performed.
Inflow rates (I, µg nutrient·m−1 RL·d−1) for macro- and
micro-nutrient elements were calculated, using the equation of
Williams [35], between harvest dates 13 and 27 days. These
values provide average accumulation rates over 14 days from
the time P-stress was imposed. The inflow equation is:
I = (N4-N1)/(T2-T1) × Ln(L4/L1)/(L4-L1)
(1)
where N4 and N1 are total macro- and micro-nutrient contents
of plants, L4 and L1 are total root length, and T2 and T1 are
time (d) for plants at harvest dates 27 and 13. Specific
accumulation rates for macro- and micro-nutrient elements
(SAcR, µg nutrient·g−1 DM·d−1) were calculated using also
the equation of Williams [35], over the same period of time
using mineral nutrient contents of shoots and shoots DM.
Data for mineral nutrient contents of plants, PEPcase,
organic acid contents, and root data (branching, density and
length) were subjected to analysis of variance using the
ANOVA procedures [29].
126
G.A. Alloush
thereafter pH values were similar in both L-P and H-P
solutions. However, the rate of OH− released from the roots of
L-P plants was smaller than that from H-P plants, the
reduction being apparent 24-h after the initiation of P-stress.
Cumulative amounts of OH− released by the roots were higher
in H-P plants, compared to L-P plants (10.43 meq·plant−1 in
H-P plants vs. 6.955 meq·plant−1 in L-P plants at day 27).
Cumulative quantities of OH− released from the roots
correlated linearly with differences between total anion-cation
taken up by the plants (R2 = 0.9987 in H-P treatment and R2 =
0.9894 in L-P treatment), the slope, however, being greater in
case of H-P plants (Fig. 4). Generally, the uptake of cation and
anions correlated linearly irrespective of P nutritional status
(Fig. 4).
Figure 1. Shoot and root dry matter production of chickpea as
influenced by high and low P supply. Bars are standard errors of
means (n = 4).
3. RESULTS
3.1. Dry matter and nutrient uptakes
Shoot and root growth was significantly reduced (P ≤ 0.01)
after 8 days from the time P-stress was imposed at day 13 from
the start of the experiment (Fig. 1). The reduction in shoot
growth (23%) was similar to the reduction in root growth
(22%), by the end of the experiment. The reduction in growth
of shoots and roots was paralleled by the reduction in the
uptake rates of NO3− and K (Fig. 2), which became apparent
by day 18. Uptake rates of Ca and P were reduced right after
the stress was imposed. The reduction of Mg and S uptake was
not apparent until day 23 (10 days from initiating P-stress).
Cumulative uptake rates of all nutrients were reduced in L-P
plants (Fig. 2), the reduction being greater for P and Ca,
compared to N, K, Mg, and S.
Both H-P and L-P plants increased the pH of the nutrient
solution (Fig. 3) throughout the entire length of the
experiment. After P-stress was introduced at day 13, the pH of
the nutrient solution in L-P plants was higher after 24-h by
about 0.5 units, compared to the H-P nutrient solution. The
higher pH in L-P solution remained until day 23, and
Average inflow rates into roots (I, µg·m−1 RL·day−1) and
specific accumulation rates into shoots (SAcR, µg·g−1
DM·day−1) of plants over the period of P-stress (between 13
and 27 days) are presented in Table I for macro- and
micronutrients. The inflow rates of all macronutrients (N, P, S,
K, Ca, and Mg) into the roots of L-P plants were reduced
compared to H-P plants. P-stress reduced significantly (P <
0.05) anion inflow rates by 17, 26, and 27% for N, P, and S
respectively, whereas the reductions in K and Mg inflow rates
(6 and 10%) were not significant. Inflow of Ca was reduced by
47%, and to a lesser extent its translocation to the shoots as
SAcR was reduced by 27%. Translocation of N, P, S, K into
shoots showed similar pattern to inflow rates and Mg SAcR
was not reduced. For B and Mo, inflow rates were reduced by
P-stress by 10 and 22% with slightly lesser reduction in their
SAcR values (13 and 17%). Mn and Fe inflows were reduced
similarly (about 18%), but their translocation to the shoots was
enhanced by 14 and 38%, whereas the inflows Zn and Cu were
enhanced by 56 and 17% and to a lesser extent, their
translocation to the shoots (17 and 13%, respectively).
The mineral composition of xylem sap collected at day 27
is shown in Table II. Concentration of all macronutrient anions
and cations in xylem sap was higher in H-P plants compared
to L-P plants and the reverse was true for micronutrients.
Irrespective of P nutritional status, total anion concentration
exceeded cation, the excess negative charge being –9.3 for
H-P plant sap vs. –3.1 for L-P plant sap.
3.2. Root morphology and characteristics
Total root length (RL) was similar in low and high P
treatments until 21 days and became significantly (P ≤ 0.05)
smaller in L-P treatment at the last harvest, 14 days after lowP treatment was imposed (Fig. 5). However, the distribution of
root branches between the different length categories was
strikingly different (Fig. 6). Almost all the lateral root
branches in the L-P root system fell in the 0–4 cm category. In
H-P plants, lateral roots were longer, up to 28–32 cm. Such
long lateral root branches were nonexistent in the L-P root
system. The density of lateral root branching along the root
system of L-P treatment was significantly greater (P ≤ 0.01)
than in H-P treatment (Fig. 7). Branching density (number of
Responses of hydroponically-grown chickpea to low P
127
Figure 2. Rate and cumulative uptake of macronutrient element uptake every 24-h in chickpea as influenced by high and low P supply.
branches·cm−1 RL) in L-P root system was almost twice that
of H-P root system. The overall average of lateral root
branches was longer in H-P root system, compared to L-P,
although average length decreased with time in both H-P and
L-P root (Fig. 7).
3.3. PEPcase activity and organic acids
The specific activity of PEP carboxylase (PEPcase) in
shoots and roots is shown in Figure 8. Enzyme PEPcase
activity, while decreasing with time towards the end of the
128
G.A. Alloush
Figure 4. Correlation of OH– released from roots and (anion-cation)
difference, and correlation between anion and cation uptakes by
chickpea after low P treatment was imposed.
Figure 3. pH values reached every 24-h in solutions, rate and
cumulative OH– released by the roots of chickpea plants as
influenced by high and low P supply.
experiment, was 10-folds higher in roots than in shoots
irrespective of P nutritional status. Both in shoots and in roots,
PEPcase activity was significantly (P ≤ 0.01) enhanced by
P-stress. The difference between L-P and H-P plants in
PEPcase activity was higher in roots than shoots.
Concentrations of organic acids in the roots decreased slightly
with time but remained almost at constant concentrations in
the shoots irrespective of P treatment (Fig. 9). Both shoots and
Figure 5. Total root length of chickpea plants as influenced by high
and low P supply. Bars are standard errors of means (n = 4).
roots of L-P plants had significantly higher organic acid
concentrations (P ≤ 0.01), compared to H-P plants during the
14 days of the treatment.
Responses of hydroponically-grown chickpea to low P
129
Figure 6. Distribution of lateral root length of chickpea as influenced
by high and low P supply. Note that the scale of L-P graph is three
times greater than the scale of H-P graph.
4. DISCUSSION
The acquisition of phosphorus by plant roots growing in
soil depends on its concentration in the soil solution and on the
buffering capacity of the soil to replenish the depleted soil
solution with P due to absorption by plant roots [24]. The
adsorption and subsequent fixation of P is high in acid and
calcareous soils, so crops may experience P deficiency later in
the growing season, even if P-fertilizers were applied before
sowing. It is therefore unlikely for soil growing plants to
experience P-free soil solution. Chickpea cultivated in the
field is normally nodulated and fixes N2, leading to
acidification of the rhizosphere, which increases the
availability of P in the soil [20]. Under the condition of the
present experiment, however, the growth of non-nodulated
chickpea plants supplied with NO3-N was reduced after 8 days
of low P supply (Fig. 1). The uptake of cations and anions
were reduced (Fig. 2), the effect being greater on anions,
compared to cations. However, anion uptake remained greater
than cation so L-P plants continued to alkaline the nutrient
solution, thus no acidification was observed (Fig. 3). In fact,
pH values were higher in L-P, compared to H-P nutrient
solution, although rates of OH− release were lower in L-P
Figure 7. Lateral roots branching density and length of chickpea
plants as influenced by high and low P supply. Bars are standard
errors of means (n = 6).
plants (Fig. 3). This is due the lower buffering capacity of the
L-P nutrient solution, where a smaller quantity of OH–
released shifted pH values higher than those recorded for H-P
plants. The quantities of OH− released from the roots did not
match the difference between anion minus cation although the
two were linearly correlated (Fig. 4). The slope, however, in
L-P treatment was lower than in H-P plants, indicating that the
rate of OH− released was lesser than expected by the
difference in anions minus cations. It is a matter of speculation
whether organic anions were actively released from the roots
of L-P plants and contributed to charge balance at the root
plasma membrane. Measurement of organic acid anion was
not performed in nutrient solutions and therefore, no firm
conclusion could be drawn. However, the lower rate of OH−
released from L-P roots coincided with greater concentrations
130
G.A. Alloush
Figure 8. Activity of PEP carboxylase in leaves and roots of chickpea
plants as influenced by high and low P supply. Bars are standard
errors of means (n = 4).
Figure 9. Concentration of organic acid anions estimated from the
alkalinity of ash in shoots and roots of chickpea plants as influenced
by high and low P supply. Bars are standard errors of means (n = 4).
of organic acid anion in the shoots and roots (Fig. 9). It is
likely that higher organic acid concentrations in the tissues
resulted from the enhanced enzyme activity of PEPcase
(Fig. 8). Accumulation of organic acids in chickpea plants was
shown to accumulate in substantial quantities in shoot and root
tissues even when no nutrient stresses were imposed [1].
Moreover, chickpea has been shown to excrete organic acid
anions into the rhizosphere and their quantities were greater
when plants were subjected to iron stress [1, 26]. However, for
tomato plants, fed with NO3-N [27], PEPcase activity
increases in the roots within 2 days of P withdrawal, by which
time the rate of alkalization of the growth medium is already
decreasing. By 8 days after withdrawal of P, PEPcase activity
in the roots can be up to four times higher than in P-sufficient
plants, and the growth medium is noticeably acidified by this
time [27]. Certainly this was not the case with chickpea grown
under condition of low P supply. The rate of OH− released was
reduced within 24 h. Chickpea did not switch to acidification,
or four-fold increase in PEPcase activity even after 14 days of
P-stress (Figs. 2, 3, 8). Acidification of the P-free nutrient
solution, even when supplied with NO3-N, was attributed to an
excess cation over anion uptake [11, 20] or the release of
organic acids (malic and citric) from the roots [10, 14].
Accumulation of organic acids in plant tissues arising from
enhanced PEPcase activity provide the source of H+ ions for
extrusion, as well as for a considerable internal anion charge,
possibly acting as counter ion in cation uptake (thus
depressing NO3− uptake) and transport from roots to
shoots [21]. Calculations of inflow and SAcR rates (Tab. I)
and analysis of xylem sap (Tab. II) support this hypothesis in
which NO3− was considerably reduced compared to cations.
Such organic acids may act as chelating agents in the soil,
exchanging with bound phosphate, and thus rendering P
available to plants [23]. Organic acids, such as citric and
malic, have been seen to increase the availability and uptake
of Fe and Mn in the soil by a chelation reaction facilitating
their movement in the soil and across roots plasma
membrane [24]. This mechanism may explain the enhanced
uptake and translocation of micronutrient cations (Zn, Cu, Fe,
Responses of hydroponically-grown chickpea to low P
131
Table I. Average inflow rates (I, mg/µg·m–1 RL·day–1) and specific accumulation rates (SAcR, mg/µg·g−1 DM·day−1) of macro- and
micronutrients in chickpea plants over 14 days of Low-P stress (between harvest 3 and 5).
Inflow rates of macronutrients (µg·m–1 RL·day−1)
N
P
S
K
Ca
Mg
H-P
211 ± 8
31 ± 2
26 ± 2
182 ± 21
109 ± 36
19 ± 1
L-P
175 ± 18
23 ± 2
19 ± 3
170 ± 16
58 ± 7
17 ± 3
Specific accumulation rates of macronutrients (µg·g–1 DM·day–1)
H-P
4763 ± 28
544 ± 33
341 ± 13
3100 ± 354
2367 ± 241
303 ± 32
L-P
3850 ± 354
412 ± 38
264 ± 29
2937 ± 157
1723 ± 228
316 ± 23
Inflow rates of micronutrients (µg·m–1 RL·day–1)
Zn
Cu
Mn
Fe
B
Mo
H-P
0.16 ± 0.01
0.06 ± 0.01
2.83 ± 0.24
3.45 ± 0.24
0.29 ± 0.02
0.09 ± 0.01
L-P
0.25 ± 0.05
0.07 ± 0.01
2.33 ± 0.46
2.80 ± 0.07
0.26 ± 0.02
0.07 ± 0.01
Specific accumulation rates of micronutrients (µg·g–1 DM·day–1)
H-P
3.24 ± 0.25
0.98 ± 0.06
21.70 ± 6.28
8.48 ± 1.11
8.14 ± 0.38
2.36 ± 0.30
L-P
3.80 ± 0.30
1.11 ± 0.06
24.73 ± 3.71
11.69 ± 1.55
7.04 ± 0.54
1.95 ± 0.19
± is standard errors of mean.
Table II. Mineral nutrient concentrations in the xylem sap of chickpea plant collected 14 days after the Low-P stress was imposed.
Treatment
Anions
NO3–
H2PO4
–†
Cations
SO4
2– †
Total A
___________________________________
–
K
+
Ca2+
Mg2+
Total C+
A-C
meq·L–1 __________________________________
H-P
9.1 ± 2.7
2.4 ± 0.4
4.6 ± 1.1
16.1
18.8 ± 4.0
4.6 ± 1.3
2.0 ± 0.4
25.4
–9.3
L-P
3.8 ± 1.1
0.6 ± 0.1
1.4 ± 0.4
5.8
7.1 ± 1.6
1.2 ± 0.4
0.6 ± 0.1
8.9
–3.1
Micronutrients (mg·L–1)
Zn
Cu
Mn
Fe
B
Mo
H-P
2.2 ± 0.7
2.1 ± 0.6
7.6 ± 1.6
5.8 ± 1.1
1.0 ± 0.5
0.2 ± 0.06
L-P
3.1 ± 0.5
3.2 ± 0.8
24.5 ± 5.0
18.0 ± 3.4
1.5 ± 0.2
0.3 ± 0.05
± is standard errors of mean.
† Calculated from total concentration of S and P, assuming all is in the form of SO42– and H2PO4–, respectively.
132
G.A. Alloush
and Mn) into the shoots of chickpea (Tab. I). The higher rate
of SAcR for micronutrient cation into the shoots of L-P
chickpea, compared to H-P plants, was also confirmed by the
higher concentrations of Zn, Cu, Fe, and Mn in the xylem sap
collected from H-P and L-P plants (Tab. II).
Chickpea has a restricted root system with fewer root
hairs [33]. It consists primarily of the main apex and
branching roots from the first and second degree lateral roots.
Root mass and total length were slightly reduced under low P
supply (Figs. 1, 5). However, morphological changes did
occur under P-stress condition. Branching density was
significantly increased in response to P-stress compared to
P-sufficient plants (Fig. 7). Most of lateral root branches were
shorter (0–4 cm), compared to lateral roots in H-P plants and
increased in number as P-stress continues (Fig. 6). This
enhanced branching and slandering may insure a greater
root surface contact with soil, and thus enhancing the
immobilization of P from soil deficient in P [4, 8]. Recently,
Malinowski et al. [22], using the same root imaging technique,
reported a decline in root diameter and increased root hair
length in endophyte infected tall fescue in response to
P deficiency, which may be one of responsible mechanisms
for the mineral stress tolerance in tall fescue. The information
about root morphological changes in P-deficient chickpea
grown in the field and under different cropping system is
lacking, and no conclusion could be made to the extent
existence of these changes under field condition. There are
however, indications to some environmental factor such as
drought, temperature, and salinity to have the reverse effect of
low-P supply on roots in which inhibits the development of
root hairs [28]. If these root morphological changes occur in
the field, they would enhance the acquisition of P and water.
This is important as chickpea is mostly grown in poor semidry areas in which the availability of fertilizers is limited.
Striking differences occur however, between chickpea
genotypes that make screening for tolerance to nutritional and
environmental stresses important [28].
These results suggest that chickpea plants respond to
P-stress in a variety of ways. They reduce alkalinization/
acidification of the rhizosphere, accumulate organic acids in
their tissues, and modify their root morphology. Modifications
of the root system occur as increase in density of lateral root
branching affecting surface area for mineral acquisition. Rootinduced changes in the rhizosphere along with the possible
release of organic acids increase P mobility in the soil. In field
conditions, chickpea is usually nodulated and acidification of
the rhizosphere may occur which could increase P
availability [24]. These responses to low P supply probably
enable chickpea to mine P from sparingly soluble sources. It is
therefore not surprising for nodulated chickpea plants grown
under field conditions to rarely exhibit P-deficiency
symptoms, even when grown in highly calcareous soils [30].
Acknowledgment: The joint support of the Fulbright Exchange Program
(CIES/USIA, No. 21133) and Tishreen University (Syria) is gratefully
appreciated. I also thank Dr. D.K. Brauer for help in the root image analyses,
and M. Smith, R. Lester, and B. Sweeney for their technical assistance.
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