New Zealand Journal of Agricultural Research

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Effects of aluminium on the root activity, organic

acids and free proline accumulation of alfalfa
grown in nutrient solution

Xiang Li Ma, Jian Ren, Wei Ran Dai, Wei Yang & Yu Fen Bi

To cite this article: Xiang Li Ma, Jian Ren, Wei Ran Dai, Wei Yang & Yu Fen Bi (2018):
Effects of aluminium on the root activity, organic acids and free proline accumulation of
alfalfa grown in nutrient solution, New Zealand Journal of Agricultural Research, DOI:
10.1080/00288233.2018.1540436

To link to this article: https://doi.org/10.1080/00288233.2018.1540436

Published online: 25 Nov 2018.

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NEW ZEALAND JOURNAL OF AGRICULTURAL RESEARCH
https://doi.org/10.1080/00288233.2018.1540436

RESEARCH ARTICLE

Effects of aluminium on the root activity, organic acids and

free proline accumulation of alfalfa grown in nutrient solution
Xiang Li Maa, Jian Rena*, Wei Ran Daia, Wei Yangb and Yu Fen Bia
a
Department of Grassland Sciences, Yunnan Agricultural University, Kunming, People’s Republic of China;
b
Kunming Cigarette Factory of Hongyunhonghe Group, Kunming, People’s Republic of China

ABSTRACT ARTICLE HISTORY

The effects of elevated aluminium (Al) concentrations on Received 18 September 2017
parameters indicating Al toxicity and Al detoxification were Accepted 23 October 2018
evaluated in two alfalfa populations from different acidic soil
KEYWORDS
environment. Plants were grown in nutrient solutions at pH 4.5, Medicago sativa; aluminium
5.5 and 6.0, with different levels of Al contamination for 30 days. toxicity; free proline; root
Shoot heights and root activity were significantly decreased in the activity; organic acids
Al treatment while free proline concentration was significantly
increased. Furthermore, citric acid and oxalic acid concentrations
tend to increase with increasing Al concentration. Different
responses were also observed between the two populations.
Shoot height, free proline concentration and root activity were
significantly higher for alfalfa plants collected from an acidic soil
environment. These plants also exhibited higher concentrations of
citric and oxalic acids when exposed to 100 μM Al at pH 4.5.
Overall, the results indicate that alfalfa plants grown on acidic
soils might develop mechanisms to be less affected by high Al
concentration.

Introduction
Alfalfa (Medicago sativa L.) has high protein and highly digestible fibre contents (Li
et al., 2010) and is one of the most important forage crops in the world with nearly
32 million hectares cultivated chiefly in temperate regions (Du et al., 2009). Alfalfa is
mainly cultivated in temperate regions, with the United States of America, Canada,
Argentina, and China accounting for about 60% of the production (Hong et al. 2009).
The increasing alfalfa consumption in the world necessitates to expand its production
area in non-traditional cultivation areas, whereas about 40% of the world’s arable
lands are acidic lands (Foy et al. 1978). Under acidic soil condition, Al3+ toxicity
might pose a serious agricultural problem (Kochian 1995). Soil pH is strongly related
to the levels of soil plant-available aluminium (Moir and Moot 2010). Especially when
the soil pH drops below 5, the octahedral hexahydrate Al(H2 O)3+ 6 , more commonly
referred to as Al3+, becomes soluble in the soil solution and might interfere with a
wide range of physical and cellular process (Kinraide 1991; Martins, et al. 2013), result-
ing in the inhibition of plant growth and functions (Giannakoula et al. 2010; Ikka et al.

CONTACT Y. F. Bi biyufenynnd@sina.com
*Second author has an equal contribution to the paper.
© 2018 The Royal Society of New Zealand
2 X. L. MA ET AL.

2013). The first reaction of plants grown on soils with elevated Al concentration was a
decreasing root growth (Foy 1988). Furthermore, the dry weight of alfalfa was signifi-
cantly reduced by Al additions, especially under acidic pH values (Langer et al. 2009).
Shoot growth of plants may be negatively affected (Larsen et al. 1997). Exposed to Al,
seedlings exhibited a reduced leaf size, and terminal buds, for example in sugar maple
(Thornton et al. 1986).
Some plant species and cultivars show tolerance to Al toxicity. The mechanism of Al
tolerance has been categorised into external or exclusion and internal detoxification
mechanism (Matsumoto 2000; Kochian et al. 2004; Kochian et al. 2005). An early
study in wheat (Triticum aestivum) reported that organic acids play a role both in Al
exclusion, via release from root and Al detoxification in the symplsam, where organic
acid such as citric acid and malic acid could chelate Al and reduce or prevent its
toxic effects at the cellular level, in particular protecting enzyme activity internally in
the plant from deleterious effect of Al (Delhaize et al., 1993). In plants like maize,
the Al-induced citric acid exudation responses are regarded as a potential Al-exclusion
mechanism (Mariano and Keltjens 2003). However, the type and amount of organic
acids released in response to Al vary widely among plant species and even cultivars
within species (Zheng et al. 1998; Kochian et al. 2004). Dong et al. (2004) observed
that soybeans exposed to Al stimulated the release of citric acid, whereas a phosphorus
(P) deficiency increased the exudation of malate and oxalate instead. Due to the impor-
tant role of organic acids in internal and external Al detoxification (Ma and Hiradate
2000), they are suitable indicators for the ability of plants to react on elevated Al
concentrations.
In addition, aluminium has also been observed to increase free proline concen-
trations under acidic condition in maize (Khan et al. 2000) and Plantago almograven-
sis and Plantago algarbiensis (Martins et al. 2013). Proline is generally assumed to
serve as a physiologically compatible solute that accumulates as needed to maintain
a favourable osmotic potential between the cell and its surroundings (Pollard and
Wyn Jones 1979; Ashraf and Foolad 2007). Proline is also involved in alleviating cyto-
solic acidosis associated with several stresses (Kurkdjian and Guern 1989), therefore,
proline accumulation can serve as a marker of plant response to stressful
environments.
Alfalfa is sensitive to Al toxicity as a result performance is limited (Dall’Agnol et al.
1996; Langer et al. 2009; Khu et al. 2010). Its yield and stand duration in acid soils are com-
promised because of inhibited root growth and reduced nitrogen fixation (Hartel and
Bouton 1989). According to Barone et al. (2008), enhanced plant growth under alu-
minium-toxic conditions can be obtained in transgenic alfalfa plants expressing citrate
synthase. However, no commercially available Al-tolerant alfalfa cultivar has been devel-
oped at present (Khu et al. 2010); there is still little information known about the mech-
anisms by which alfalfa responds to and resist Al stress. In our study, two populations of
alfalfa that adapted to different soil pH value, respectively, were employed as experimental
materials (Table 1). The aim of this investigation was to (a) determine root activity, free
proline and organic acid accumulation as affected by Al rhizotoxicity; (b) evaluate
whether alfalfa plants grown on acidic soils develop mechanisms to tolerate high Al
concentration.
 NEW ZEALAND JOURNAL OF AGRICULTURAL RESEARCH 3

Table 1. The origin of the investigated two alfalfa populations and soil conditions from the collection
area.
Longitude Latitude Altitude
No. (N) (E) (m) Soil pH Soil type
Dali 25°32′ 21.3′′ 100°19′ 15.0′′ 2,012 8.37 Grey clay loam
Tonghai 24°05′ 29.1′′ 102°46′ 25.0′′ 1890 4.46 Red soil

Materials and methods

Plant material and growth conditions
Two alfalfa populations–Dali and Tonghai were used in this study (Table 1). Seeds were
collected from the Yunnan-Guizhou Plateau, southwestern China. After surface sterilisa-
tion of the seeds with HgCl2 (0.1%), they were put on filter paper saturated with distilled
water in Petri dishes and incubated in a growth chamber at 25°C for 4 days. Then, seed-
lings were selected for uniformity and then transplanted to polypropylene pots containing
the nutrient solution (Hoagland and Arnon 1959). The nutrient solutions consisted of
2.5 mM KNO3, 2.5 mM Ca(NO3)2, 1 mM MgSO4, 0.5 mM KH2PO4, 0.04 mM Fe
(III)EDTA, 0.009 mM MnCl2·4H2O, 0.4 μM ZnSO4·7H2O, 0.3 μM CuSO4·5H2O,
0.0451 mM H3BO3, 0.072 μM NH4Mo 5H2O. Nutrient solutions were continuously
aerated with pumps and renewed every 5 days. Three Al levels (0, 50 and 100 μM)
added in the form of aluminium chloride (AlCl3) and three levels of pH (4.5, 5.5 and
6.0) were tested. The pH was kept constant by daily additions of 0.1 M hydrochloric
acid (HCl) or sodium hydroxide (NaOH) as required. The experiment was conducted
in a controlled-environment growth chamber for 30 d at 25°C with a 60 to 75% relative
humidity and a 12 h light photoperiod.

Measurement of shoot height and free proline concentration

Plant growth was expressed as shoot height, measured after 30 days of Al treatment.
Free proline was measured as described by Bates et al. (1973). The fully expanded and
exposed leaves (0.2 g) were homogenised in 5 ml of 3% sulphosalicylic acid solution. After
centrifugation, 2 ml supernatant, 2 ml glacial acetic acid and 2 ml 2.5% acid ninhydrin sol-
ution were added into a test tube covered with a Teflon cap. The absorbance of the free
proline concentration was measured at 520 nm. The proline concentration was expressed
as mmol g−1 fresh weight (FW).

Determination of the root activity

Root activity was quantified by measuring root dehydrogenase activity using triphenylte-
trazolium chloride (TTC) reduction technique (Joslin and Henderson 1984), which is
related positively to respiration capacity of different tissues like root. To determine the
amount of live or dead roots in a sample, roots were first washed with deionised water,
and fresh weight was determined. Roots then were placed into test tubes with 0.4% (w/
v) TTC in 0.06 M Na2HPO4–KH2PO4 (pH 7.0) and tubes were incubated in the dark at
37°C water bath for 1 h. Formazan a water-insoluble red substance, which was formed
from the reduction of TTC by dehydrogenase enzymes in living tissues, was extracted
4 X. L. MA ET AL.

in ethyl acetate. The absorbance of the extract was recorded at 485 nm with a spectropho-
tometer (Shimadzu Scientific Instrument, Columbia, MD, USA). A regression of absor-
bance against root weight was determined for live roots to calculate the percentage of
live root tissue among all roots in dry weight, which is considered the root activity.

Determination of organic acid

After 30 days of growth, shoots, leaves and roots were harvested and washed by deionised
water. After mixing, shoots, leaves and roots (0.25 g) were ground in 0.5 M HCl, then sol-
utions were bathed boiled water 20 min. In order to quantify the concentration of organic
acids (oxalic, citric and malic acid), extracts were lyophilised and the residue re-dissolved
in 5 ml of deionised water, after filtration (0.45 μm) HPLC analysis was carried out. Sep-
aration was achieved on a 250 × 4 mm reverse phase column (Merck, LiChrospher 100
RP-18, 5-μm particle size). Sample solutions (10 μL) were injected into the column, and
a 5%CH3OH-0.1 ml KH2PO4 (pH 2.7) solution was used for isocratic elution at a flow
rate of 0.8 ml min−1 with UV detection at 214 nm. Preliminary studies with standard
organic acids indicated that recovery of the organic anions was about 98%. The concen-
tration of organic acids was analysed by HPLC referred to Ma et al. (1997).

Experimental design and data analysis

The experimental design was a randomised complete block with three replications. Stat-
istical analyses were performed using the statistical software package for social science
(SPSS) version 13. Two-way analyses of variance (ANOVA) were used to test the main
effects of the Al treatment, populations and their interaction. Within each population,
pairwise comparisons were made using the Student-Newman-Keuls multiple range
tests. Differences were considered significant at the P < .05 level.

Results
Effect of Al on shoot height
For both populations, shoot heights were significantly decreased by 50 and 100 μM Al
treatment, whereas a stronger inhibition was observed in the 100 µM Al treatment
(Figure 1). After 30 days, the growth was almost completely inhibited under the highest
Al concentration. Also, the shoot height between the two populations differed signifi-
cantly. Compared to Tonghai, shoot height under Al stress was about 39% lower in
Dali. Moreover, the interaction between pH and Al has a significant effect on shoot
height of Dali. A further decrease was observed when plants were treated by the highest
Al and the lowest pH.

Effect of Al on free proline concentration

When plants were exposed to 50 and 100 µM Al, the free proline concentration was sig-
nificantly increased in both alfalfa populations (P < .001) (Figure 2), particularly at pH 4.5.
However, with the increase of pH, less free proline was accumulated among all the
 NEW ZEALAND JOURNAL OF AGRICULTURAL RESEARCH 5

Figure 1. Shoot height of two alfalfa populations–Dali and Tonghai exposed to different aluminium
concentrations and pH values. Each value is a mean ± SE. Different lower case letters denote statistically
significant differences between the Al treatments (P .05) according to the Student-Newman-Keuls mul-
tiple range test. The results of analysis of variance are abbreviated as follows: Al = effect of the Al treat-
ment; pH = effect of the pH treatment; and Al × pH = Al ×pH interaction effect.

investigated material. Furthermore, the magnitude of the response differed between pH

values. At pH 4.5, free proline of Tonghai population in the 100 µM Al treatment accumu-
lated to almost twice the amount of treatment without Al, whereas such responses were
not observed at pH 6.5. The effect of Al toxicity on the free proline concentration was
stronger in the Tonghai population compared to the Dali population.

Effect of Al on root activity

For both populations, root activity decreased in parallel with the decrease of pH values. In
the Dali population, root activity at pH 4.5 was significantly decreased in the 100 μM Al
treatment (P < .05), but not in the 50 μM Al treatment. At all pH level, Tonghai showed
the highest root activity (Figure 3). In the 100 μM Al treatment at pH 4.5, the root activity
6 X. L. MA ET AL.

Figure 2. Free proline concentrations of two alfalfa populations–Dali and Tonghai exposed to different
aluminium concentrations and pH values. Each value is a mean ± SE. Different lower case letters denote
statistically significant differences between the Al treatments (P < .05) according to the Student-Newman-
Keuls multiple range test. The results of analysis of variance are abbreviated as follows: Al = effect of the
Al treatment; pH = effect of the pH treatment; and Al × pH = Al × pH interaction effect.

of Tonghai population was 246% significantly higher than the root activity of the Dali
population (P < .001).

Effect of Al on organic acid accumulation

For both populations, citric acid contents were significantly affected by the Al concen-
tration and the pH value; the citric acid content in the plants was significantly increased
when exposed to 100 µM Al at pH 5.5 or 6.5 (Table 2). However, a significant difference
was observed between the two populations. The citric acid content under Al stress in the
Tonghai population was significantly higher than that in the Dali population (P < .001).
Moreover, at pH 4.5, a concentration of 100 μM Al significantly increased the citric
acid content in the Tonghai population by 90% compared to the control, whereas such
effect was not observed in the Dali population.
 NEW ZEALAND JOURNAL OF AGRICULTURAL RESEARCH 7

Figure 3. Root activity of the two alfalfa populations–Dali and Tonghai exposed to different aluminium
concentrations and pH values. Each value is a mean ± SE. Different lower case letters denote statistically
significant differences between the Al treatments (P < .05) according to the Student-Newman-Keuls
multiple range test. The results of analysis of variance are abbreviated as follows: Al = effect of the
Al treatment; pH = effect of the pH treatment; and Al × pH = Al × pH interaction effect.

The oxalic acid concentration increased significantly in response to increasing Al con-

centrations; this effect was stronger at low pH values. At pH 4.5, the oxalic acid in the
Tonghai population was three times higher than the control when exposed to 100 μM
Al concentration; In contrast, the oxalic acid content in Dali was just increased by 40%
compared to the control. However, the magnitude of increase depended on the alfalfa
population, with concentrations in Tonghai population being significantly higher than
in Dali population (P < .001).
Malic acid concentration was significantly affected by the pH value as well as the Al
treatment. In Dali, malic acid tended to increase with increasing Al concentrations at
pH 5.5 and 6.5, but not at pH 4.5. Particularly at pH 4.5, the malic acid concentration
was significantly decreased by the 100 µM Al treatment while being increased by the
50 µM Al treatment. In addition, no significant difference in malic acid concentration
was found between Tonghai and Dali populations.
8 X. L. MA ET AL.

Table 2. Citric acid, oxalic acid and malic acid concentrations in the plants of the alfalfa populations–
Dali and Tonghai grown in nutrient solutions with different pH values and aluminium concentrations.
Alfalfa Al treatment Citric acid
population pH value (μM) (μmol h −1gFW−1) Oxalic acid (μmol h −1gFW−1) Malic acid (μmol h −1gFW−1)
Dali pH 4.5 Al 0 1.10 ± 0.001 b 1.04 ± 0.001 c 1.03 ± 0.001b
Al 50 1.76 ± 0.002 a 1.19 ± 0.001 b 2.49 ± 0.001a
Al 100 0.92 ± 0.001 c 1.46 ± 0.001 a 0.92 ± 0.003c
pH 5.5 Al 0 0.88 ± 0.02 c 0.92 ± 0.001 c 1.2 ± 0.003 c
Al 50 1.44 ± 0.001 b 1.03 ± 0.017 b 1.98 ± 0.002 b
Al 100 1.55 ± 0.003 a 1.22 ± 0.002 a 3.93 ± 0.001 a
pH 6.5 Al 0 0.90 ± 0.002 c 0.76 ± 0.001 c 0.84 ± 0.001 c
Al 50 0.93 ± 0.001 b 0.94 ± 0.001 b 0.87 ± 0.003 b
Al 100 0.98 ± 0.001 a 1.35 ± 0.001 a 1.09 ± 0.001 a
P > FAl 0.000 0.000 0.000
P > FpH 0.000 0.000 0.000
P > FAl × pH 0.000 0.000 0.000
Tonghai pH 4.5 Al 0 1.25 ± 0.002 b 1.64 ± 0.002 c 1.77 ± 0.001 c
Al 50 1.23 ± 0.001 c 2.50 ± 0.001 b 1.93 ± 0.001 b
Al 100 2.38 ± 0.003 a 4.93 ± 0.001 a 2.12 ± 0.001 a
pH 5.5 Al 0 1.16 ± 0.001 b 1.4 ± 0.002 c 1.53 ± 0.001 b
Al 50 1.05 ± 0.002 c 2.22 ± 0.001 b 1.70 ± 0.002 a
Al 100 2.96 ± 0.003 a 2.46 ± 0.001 a 1.25 ± 0.001 c
pH 6.5 Al 0 1.01 ± 0.001 c 1.21 ± 0.001 c 1.27 ± 0.002 a
Al 50 2.47 ± 0.002 a 1.94 ± 0.001 b 0.53 ± 0.003 c
Al 100 2.33 ± 0.001 b 3.91 ± 0.001 a 1.25 ± 0.001 b
P > FAl 0.000 0.000 0.000
P > FpH 0.000 0.000 0.000
P > F Al × pH 0.000 0.000 0.000
P > FP 0.000 0.000 0.521
P > FpH 0.922 0.093 0.000
P > FP × pH 0.185 0.325 0.006
Each value is a mean ± SE. Lower case letter denotes statistically significant differences between the Al treatments (P <
0.05) according to the Student-Newman-Keuls multiple range test. The results of analysis of variance are abbreviated
with: FAl = Al effect; FpH = pH effect; FP = population effect; FAl × pH = Al × pH interaction effect. F(P × pH) = Popoluation ×
pH interaction effect.

Discussion
Aluminium has long been believed to be one of the major factors that limiting the world-
wide crop production. Al has been a potential threat to affect legume forge production
such as alfalfa in the world. Our results indicated that shoot height, free proline concen-
tration, root activity as well as tissue concentrations of organic acids of M. Sativa growing
in nutrient solutions were significantly affected by concentrations of 50 and 100 μM Al at
different pH levels compared to plants grown without Al treatment,
The growth of the plant is widely studied concerned the toxicity of Al, thereat most
investigations targeted on roots as this is the directly affected organ. It is found that
exposure of root apex to Al causes a rapid inhibition of root growth (Ryan et al. 1993;
Langer et al. 2009) as a result of Al-induced inhibition of H+-ATPase activity (Ahn
et al. 2002), and depolarisation of the membrane (Pavlovkin et al. 2009). However, the
suppressed effect on root depended on the legume species (Berenji et al. 2017).
Studies conducted so far indicate that there is little knowledge about Al effects on the
aboveground parts (shoots and leaves) of plants. In our study, shoot growth was signifi-
cantly inhibited by Al, especially at a pH value of 4.5. According to Rengel (1996), the
effects of Al3+ toxicity on shoots, such as restricted growth, become evident only after
root growth is limited by exposure to toxic Al3+ levels in the rhizosphere, leading to
 NEW ZEALAND JOURNAL OF AGRICULTURAL RESEARCH 9

mineral nutrition deficiencies in the aboveground tissues. Thus, the reduction in shoot
growth and crop yield due to Al toxicity seems to be a long-term effect (Taylor 1988).
In our study, we could show that the shoot height under Al stress differs significantly
for different alfalfa populations; the Tonghai population which was collected from more
acidic soil pH values (4.46) exhibited a larger shoot height than the Dali population col-
lected from soils with a higher pH value. Also, Langer et al. (2009) found that different
alfalfa cultivars are significantly different affected in terms of shoot biomass under Al
stress. Therefore, the magnitude of growth reduction depended on the alfalfa population.
Similar results were also reported in high bush blueberry by Reyes-Díaz et al. (2010) who
investigated the effects of Al in nutrient solutions on relative growth rate. Moreover,
different tolerance to Al toxicity between species was found in lotus and falcate lucerne
(Moir et al. 2016).
In term of free proline concentrations, they were significantly increased in the exper-
imental population in response to 50 and 100 µM Al. However, it was found that free
proline concentration was more influenced by 100 µM Al than by 50 µM Al treatments.
According to Giannakoula et al. (2008; 2010) and Khan et al. (2000), more increase of
free proline was related to aluminium tolerance of plants. Increase in proline level suggests
that anti-stress mechanism of alfalfa is efficient to control changes induced by aluminium,
as also found by Yang and Cheng (2001) in Mungbean as well as in P. almogravensis and
P. algarbiensis (Martins et al. 2013).
Root activity brings about effective nutrients absorption at the root for growth and yield
of crops. In our study root activity of alfalfa was significantly affected by Al treatment, par-
ticularly at pH 4.5. Root activity is not only related to the aerobic respiration of roots but
also associated with dehydrogenase activity of tricarboxylic acid (TCA) cycle that regulates
sugar and mineral absorption in plants (Onanuga et al. 2012). According to the study of
Atkin et al. (2000), a decrease in the rate of root growth lowers the overall requirements for
respiratory ATP, thus the rate of total respiration. The decrease of root activity might attri-
bute to the inhibition of root growth.
Many studies focused on the exudations of organic acid (Zheng et al. 1998; Dong et al.
2004; Langer et al. 2009). Organic acids like citric acid, oxalic acid and malic acid are con-
sidered to effectively detoxify Al (Kochian et al. 2004), and their content has been corre-
lated with aluminium tolerance in higher plants. On the other hand, Takeda et al. (1985)
and Langer et al. (2009) found that Al can accumulate to very high levels in shoot or root,
leaves, for example in leaves of Buckwheat, the Al concentration was as high as 15,000 µg
Al g−1 dry weight when the plant was grown on acid soils (Ma and Hiradate 2000). There-
fore, organic acids with Al-chelating ability play an important role in the detoxification of
Al both externally and internally (Ma and Hiradate 2000). It is equally important to learn
the accumulation conditions of organic acid in plants. According to De la Fuente et al.
(1997), over production of citric acid was shown to result in aluminium tolerance in
plants like transgenic tobacco (Nicotiana tabacum) and papaya (Carica papaya).
Enhanced plant growth under acidic, aluminium-toxic conditions can be obtained in
transgenic alfalfa plants expressing citrate synthase Barone et al. (2008), Moreover, the
citrate content of alfalfa and Eucalyptus increased with increasing Al application
(Langer et al. 2009; Ikka et al. 2013). Our results are in agreement with these findings.
At the same time, in our study, the content of citric and oxalic acid was much higher
in Tonghai than in Dali population (P < .001) and increased in the presence of Al much
10 X. L. MA ET AL.

more in the former than in the latter, suggesting that the increased content of these organic
acids contributed to the Al resistance of Tonghai population. Role of organic acid to detox-
ify Al is different. Ma et al. (1997) and Shen et al. (2002) showed that most of the Al in
plants of buckwheat was complexed with oxalate in a 1:3 Al-oxalate complex. Later,
they found that al being transported to the shoot is complexed with citrate, not oxalate
(Ma and Hiradate 2000). Based on the study of Andrade et al. (2011), the Al-sensitive
wheat cultivar had a higher concentration of malic acid regardless of the presence of
Al. Considered no significant difference in the concentration of malic acid was found
between Tonghai and Dali populations, hence, we believe that citric acid is much more
effective at detoxifying Al in alfalfa than malic acid.

Conclusions
In our study, some growth and physiological properties of alfalfa were greatly affected by
increases of Al content in growing solutions. The observed differences between the study
populations in shoot height, free accumulation and root activity as well as content of citric,
oxalic acid indicate the Tonghai population is more tolerant to Al stress than the Dali
population. Therefore, we concluded that alfalfa plants grown on acidic soils might
develop mechanisms to be less affected by high Al concentration.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The research was supported by the National Natural Science Foundation of China [grant number
31601997; grant number 30860198].

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