Environmental and Experimental Botany 71 (2011) 114–120

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Environmental and Experimental Botany

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

Stimulation of root acid phosphatase by phosphorus deficiency is regulated by

ethylene in Medicago falcata
Yan-Su Li a , Yan Gao a,b , Qiu-Ying Tian a , Feng-Ling Shi b,∗∗ , Ling-Hao Li a , Wen-Hao Zhang a,∗
a
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, PR China
b
College of Ecology and Environmental Science, Inner Mongolia Agricultural University, Huhehot 010018, PR China

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

Article history: Plants have developed numerous strategies to cope with phosphorus (P) deficiency resulting from low
Received 1 July 2010 availability in soils. Evolution of ethylene and up-regulation of root secreted acid phosphatase activity
Received in revised form 9 November 2010 are common for plants in response to P deficiency. To determine the role of ethylene in response of plants
Accepted 14 November 2010
to P deficiency, we investigated the effects of ethylene precursor (1-amino cyclopropane-1-carboxylic
acid, ACC) and ethylene synthesis antagonists (aminoethoxyvinylglycine AVG, cobalt, Co2+ ) on P con-
Keywords:
centrations in roots and shoots of Medicago falcata seedlings grown in P-sufficient (500 ␮M H2 PO4 − ) and
Phosphorus deficiency
P-deficient (5 ␮M H2 PO4 − ) solution. After transferring M. falcata seedlings from P-sufficient to P-deficient
Ethylene
Medicago falcata
solution for 2 days, root P concentration was significantly reduced. The reduction in root P concentra-
Acid phosphatase activity tion was reversed by AVG and Co2+ , and a similar reduction in root P concentration of seedlings exposed
Phosphate transporters to P-sufficient solution was observed by ACC. Expression of high-affinity phosphate transporters (MfPT1,
Organic phosphorus MfPT5) was enhanced by P-deficiency and this process was reversed by AVG and Co2+ . There was a marked
increase in activity of root acid phosphatase (APase) and expression of gene encoding APase (MfPAP1)
under P-deficient conditions, and the increase in APAse activity and expression of MfPAP1 was inhibited
by AVG and Co2+ . APase activity and expression of MfPAP1 expression in seedlings grown in P-sufficient
solution were enhanced by ACC. Root and shoot P concentrations were increased when organic phospho-
rus was added to the P-deficient solution, and the increase in P concentration was significantly inhibited
by AVG and Co2+ . These results indicate that ethylene plays an important role in modulation of P acqui-
sition by possibly mobilizing organic P via up-regulating root APase activity and high-affinity phosphate
transporters.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction Plants have evolved numerous mechanisms to adapt to P-

Phosphorus (P) is a critical macronutrient required for numer-
ous functions in plants and is one of the limiting factors for plant
growth due to its rapid immobilization by soil organic and inor-
ganic components (for review, see Vance et al., 2003; Richardson
et al., 2009). The application of P fertilizer to cropland has become
a routine agricultural practice worldwide. However, the use of P
fertilizer is not only expensive but is also polluting environment
and non-sustainable. Thus, it is imperative to elucidate the mecha-
nisms by which plants respond and adapt to the P-deficient growth
medium.
∗ Corresponding author at: State Key Laboratory of Vegetation and Environmental
Change, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, PR
China. Tel.: +86 10 6283 6697l; fax: +86 10 6259 2430.
∗∗ Corresponding author. Tel.: +86 471 4301371; fax: +86 471 4300732.
E-mail addresses: sfl0000@126.com (F.-L. Shi), whzhang@ibcas.ac.cn
(W.-H. Zhang).
deficient soils. These include changes in root architecture to
maximize surface area for uptake of P with the extreme exam-
ple being the development of specialized “cluster root” in certain
species (Shane and Lambers, 2005), acidification of root rhizo-
sphere and exudation of organic anions to solubilize P that is bound
with cations and other mineral ligands in the soil (Ryan et al., 2001),
and secretion and expression of acid phosphatases from roots to
hydrolyze soil organic P (George et al., 2005; Tran et al., 2010).
In soils, up to 80% of total P occurs as organic P that comprises
mainly monoesters (Richardson et al., 2009). Although the overall
significance of soil organic P in plant nutrition remains unknown,
it has been shown that plants can use several P sources in both
sand and soil culture (Duff et al., 1994; Li et al., 2003). Soil organic
P has to be hydrolyzed to inorganic P that is available to plants
by phosphatases prior to acquisition by plants. Acid phosphatases
(APase, EC 3.1.3.2) catalyze the hydrolysis of P from a broad and
overlapping range of P-monoesters with an acidic pH optimum
(Duff et al., 1994). A number of studies have reported that secretion
and expression of APase are enhanced under P-deficient conditions

0098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2010.11.007
 Y.-S. Li et al. / Environmental and Experimental Botany 71 (2011) 114–120
(Tadano et al., 1993; Miller et al., 2001; Wasaki et al., 2003; Playsted
et al., 2006; Wasaki et al., 2009). The enhanced activities of APases
facilitate to hydrolyze monoester soil organic P at low pH, thus
increasing orthophosphate availability. The physiological function
of root APases has been confirmed by that transgenic plants over-
expressing APase genes exhibit greater P acquisition with organic
P as the sole P resource than wild-type plants (Xiao et al., 2006;
Wang et al., 2009; Wasaki et al., 2009). The librated inorganic P
is taken up by plants via phosphate transporters located in cell
membranes of root cells. In this context, it has been shown that
up-regulation of several high-affinity phosphate transporters is a
common phenomenon in response to P deficiency (Raghothama,
1999).
Ethylene, also referred to as “stress hormone”, is closely associ-
ated with nutrient deficiencies and toxicities (Lynch and Brown,
1997; Schmidt, 2001). For instance, it has been reported that P
deficiency evokes ethylene production (Borch et al., 1999; Lynch
and Brown, 2001; Schmidt, 2001; Li et al., 2009). The P-deficiency-
induced ethylene production and/or changes in sensitivity to
ethylene have been shown to be involved in regulation of root
architecture (Borch et al., 1999; Ma et al., 2003; Zhang et al., 2003;
He et al., 2005; Kim et al., 2008) and root hydraulic conductivity
(Li et al., 2009). A recent study reported that ethylene regulates
phosphorus remobilization and expression of a phosphate trans-
porter gene (PhPT1) during petunia corolla senescence (Chapin and
Jones, 2009). However, there has been no study to examine the
role of ethylene in P acquisition by plants in response to P defi-
ciency. To test whether ethylene is involved in processes associated
with P acquisition under P-deficient conditions, we investigated the
role of ethylene in regulation of APase activity, phosphate trans-
porters and P uptake in roots under short-term P deficiency using
legume plants of Medicago falcata, a native legume plant widely
distributed in Inner Mongolia grassland in northern China. Our
results revealed that P-deficiency-induced ethylene evolution was
involved in mobilization of organic P by modulating APase activity
and phosphate transporters.
2. Materials and methods
2.1. Plant material
Seeds of M. falcata L. (ecotype Ximeng), which were obtained
from the Institute of Grassland Research, the Chinese Academy
of Agricultural Sciences, were dipped in sulfuric acid for approx.
5 min to destroy the seed capsule, and then rinsed thoroughly
with sterilized water. Thereafter the seeds were spread on 0.75%
agar to germinate at 25 ◦ C till the radicles being about 2 cm.
The seedlings were transferred to plastic buckets filled with
5 L of fully aerated nutrient solution (Barker et al., 2006). Each
bucket contained 15 plants. Plants were grown in a growth
chamber with 25 ◦ C (day)/20 ◦ C (night), 80% relative air humid-
ity, 200–230 ␮mol m−2 s−2 photosynthetic photon flux density and
16/8 h day–night period. After grown in the culture solution for
3 weeks, half of the plants were transferred to P-deficient (−P)
solutions. Potassium phosphate (KH2 PO4 ) was added to give a
final concentration of 500 ␮M of H2 PO4 − for P-sufficient (+P) and
5 ␮M for P-deficient (−P) media, respectively. To maintain the
identical concentrations of K between P-sufficient and P-deficient
solutions, 455 ␮M KCl was added in the P-deficient solutions. The
composition of other nutrients in the hydroponic nutrient solution
was: 1 mM MgSO4 , 0.25 mM K2 SO4 , 0.25 mM CaCl2 , 1 mM NH4 NO3 ,
2.5 mM KNO3 , 100 ␮M Fe-Na-EDTA, 30 ␮M H3 BO3 , 5 ␮M MnSO4 ,
1 ␮M ZnSO4 , 1 ␮M CuSO4 , 0.7 ␮M Na2 MoO4 and 50 ␮M KCl. pH of
the hydroponic solution was adjusted to 6.0. To determine the con-
tribution of organic P to the total P content in roots and shoots,
inositol phosphate (phytic acid) at 83.3 ␮M, which led to the total
115
organic P concentrations of 500 ␮M in the solution, was added into
the +P and −P solutions, respectively.
2.2. Measurements of acid phosphatase activity of root
The acid phosphatase activity of roots was assayed following
protocols used by Playsted et al. (2006) with some minor modifi-
cation. The 3-week-old M. falcata seedlings were transferred from
+P solutions to filter-sterilized +P and −P solutions in the absence
and presence of ethylene synthesis inhibitors (0.5 ␮M AVG, 10 ␮M
Co2+ ) and ethylene precursor (1 ␮M ACC). After incubation in the
+P and −P solution with varying chemicals for 2 days, roots were
excised and rinsed thoroughly with sterile deionized water. For the
treatment with 1-MCP, P-deficient M. falcata roots were treated
with 0.1 g mL−1 1-MCP in an air-tight container for 2 days. The
excised roots were incubated in 10-mL sterile solution containing
10 mM p-nitrophenyl phosphate (p-NP), 0.5 mM CaCl2 and 15 mM
2-[N-Morpholino] ethanesulfonic acid (MES) (pH 5.8) at 27 ◦ C for
60 min under dark. The reaction was terminated by the addition of
250 mM NaOH, and acid phosphatase activity was determined in a
spectrophotometer at 412 nm relative to standard solutions of p-
NP. Phosphatase activity was expressed as mmole of 4-nitrophenol
released per hour per gram of root dry weight (mmol p-NP h−1 root
DW−1 ).
2.3. Expression patterns of genes encoding P transporters and
acid phosphatase
Total RNAs were extracted from M. falcata roots with Trizol
reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-
free DNase I (Promega, Madison, WI, USA). Prior to cDNA synthesis,
PCR was carried out with each of the extracted RNA samples as
templates and selected primers to validate that these RNA samples
were free of DNA. cDNA synthesis was carried out using a Super-
script II synthesis kit (Invitrogen, Carlsbad, CA, USA). Gene-specific
primers were designed using Primer Express Software Version 3.0
(Applied Biosystems, Warrington, UK). qRT-PCRs were performed
using a Mx3000PTM Real-Time PCR System (Agilent Technology,
Santa Clara, CA, USA). The cycling conditions were 7 min at 95 ◦ C,
followed by 40 cycles of 95 ◦ C for 10 s, and 60 ◦ C for 30 s. The fol-
lowing four primer pair combinations were used in the qRT-PCRs
were:
MfPT1 5-TACCTCCGGCAAAAGAAATGAATG-3 and 5-
TAAATGCTACCGTGAACCAGTAACCC-3,
MfPT5 5-ACCCAACTGGTATTGGTATCAAGAACTCC-3 and
5-CATCTCAATAGCCTCAGCATCGTCATC-3,
MfPAP1 5-TGGTGTTTGTGTGTAATGGAGGCAGA-3 and 5-
CCTTGTGTTATATGAACCTGTTGGGGAG-3, and
MfPHY1 5-TATTATTCTTTCAATGCGGGAGGA-3 and 5-
GACTTGTAAGTGCTGTACCAAGGTGC-3.
In addition, a housekeeping gene, MfActin, was employed as a
control: 5-ACGAGCGTTTCAGATG-3 and 5-ACCTCCGATCCAGACA-3.
The primers were designed based on the cDNA sequence of AtPAP,
AtPT1, AtPT5 and AtActin. Primers were designed across exon–exon
junctions of cDNA to avoid potential problems due to contami-
nating genomic DNA. Amplification efficiency for each primer pair
was calculated using serial cDNA dilutions, the expression values
of the four genes were compared with the MfActin. Two technical
replicates were performed for every biological repeat.
2.4. Determination of P concentration in roots and shoots
After M. falcata seedlings were grown in P-sufficient solution for
3 weeks, they were exposed to P-deficient and P-sufficient solu-
tions containing varying chemicals (1 ␮M ACC, 0.5 ␮M AVG, 10 ␮M
CoCl2 ) in the absence and presence of 83.3 ␮M inositol phosphate
116 Y.-S. Li et al. / Environmental and Experimental Botany 71 (2011) 114–120

Fig. 1. Effect of P deficiency (−P), ACC (1 ␮M), AVG (0.5 ␮M) and Co2+ (10 ␮M) on P concentrations in roots (A and B) and shoots (C and D) in M. falcata seedlings pre-incubated
in P-sufficient (+P) for 3 weeks. The seedlings were incubated in solutions containing different chemicals for 48 h and P concentrations in roots and shoots were determined.
Data are mean ± SE with at least three replicates for each treatment and bars with different letters indicate significant difference at P < 0.05.

for 2 days. Thereafter roots and leaves from both P-sufficient and P-
deficient seedlings exposed to varying treatments were harvested,
and dried at 75 ◦ C for 2 days after washed thoroughly with deion-
ized water. Following perchloric/nitric acid digestion, phosphorus
concentration was determined by ICP-OES (Varian 700, USA).
2.5. Statistical analysis
The analysis of variance was conducted between different
treatments. The significant differences between treatments were
evaluated by LSD multiple range tests (P < 0.05) using the SAS sta-
tistical software.
3. Results
evolution plays a role in mediating P acquisition in roots under P-
deficient conditions. To test this hypothesis, we further analyzed
the response of P transporters to deficiency and ethylene under P-
sufficient and P-deficient conditions at transcriptional level. Two
genes encoding the high-affinity P transporters in M. falcata (MfPT1
and MfPT5) has been identified (Liu et al., 2008) and were found
to be up-regulated when exposed to P-deficient solution (Fig. 2).
The P-deficiency-induced increases in MfPT1 and MfPT5 transcripts
were substantially attenuated by AVG and Co2+ , with AVG being
more effective than Co2+ (Fig. 2). A similar increase in MfPT1 and
MfPT5 transcripts was also observed when P-sufficient seedlings
were treated with ACC (Fig. 2). These findings imply that ethylene
is likely to be involved in P-deficiency-induced up-regulation of the
high-affinity phosphate transporters at transcriptional level.

3.1. Effect of P-deficiency on P accumulation in root and shoot
There was a sharp reduction in P concentration in roots when
M. falcata seedlings grown in P-sufficient solution were transferred
to P-deficient solution for 48 h (Fig. 1A and B). In contrast to roots,
P concentration in shoots of M. falcata seedlings was not affected
when exposed to the same P-deficient solution for 48 h (Fig. 1C and
D). Our previous study demonstrated P deficiency evoked a marked
evolution of ethylene from M. falcata roots (Li et al., 2009). To test
whether the P-deficiency-induced ethylene may play a role in reg-
ulation of the reduction in root P concentration, we investigated
the effect of ethylene precursor ACC, and antagonists of ethylene
synthesis AVG and Co2+ (Lau and Yang, 1976) on P concentrations
in roots and shoots of P-sufficient and P-deficient seedlings. No
effects of ACC and AVG on P concentrations in shoots (Fig. 1C and
D). In contrast, treatment with ACC led to a significant reduction
in root P concentration of P-sufficient seedlings (Fig. 1A and B). On
the other hand, the P-deficiency-induced reduction in root P con-
centration was substantially alleviated by AVG and Co2+ (Fig. 1A
and B). These results suggest that P-deficiency-induced ethylene
3.2. Phosphorus deficiency-induced increase in APase activity
was mimicked by ethylene
To test whether P deficiency stimulates APase from M. fal-
cata roots, effect of P deficiency on APase activity from M. falcata
root exudates was studied. As shown in Fig. 3, root APase activ-
ity was markedly enhanced upon exposure of M. falcata seedlings
to P-deficient medium (Fig. 3). For instance, the APase activ-
ity was increased from 1.23 ± 0.13 to 6.47 ± 0.28 mmol p-NP g−1
root DW h−1 after exposure to P-deficient solution for 48 h. Our
previous studies have shown that P-deficiency-induced ethylene
evolution from roots of the same plant species as used in the
present study (Li et al., 2009). To establish the link between the
P-deficiency-induced ethylene and the increase in APase activity,
we first examined the effect of AVG and Co2+ on APase activity
of P-deficient roots. Inhibition of ethylene evolution by AVG and
Co2+ markedly reduced the P-deficiency-induced increase in APase
activity (Fig. 3). In addition to inhibition of ethylene biosynthe-
sis, antagonist of ethylene action 1-MCP was also used to block
the ethylene action. As shown in Fig. 3B, the P-deficiency-induced
 Y.-S. Li et al. / Environmental and Experimental Botany 71 (2011) 114–120 117

Fig. 3. Changes in activities of acid phosphatase in response of 3-week-old M. falcata

Fig. 2. Effect of ACC, AVG and Co2+ on expression of phosphate transporters (MfPT1
and MfPT5) in M. falcata seedlings incubated in P-sufficient (+P) and P-deficient (−P)
solution. M. falcata seedlings were treated in +P and −P solution with 1 ␮M ACC,
0.5 ␮M AVG and 10 ␮M Co2+ for 6 h. Relative mRNA level determined by qRT-PCR and
was normalized based on the mRNA in roots grown +P solutions. Data are mean ± SE
of three replicates and bars with different letters indicate significant difference at
P < 0.05.
seedlings grown in P-sufficient solution (+P) in response to treatments with 48-h
P deficiency (−P), 1 ␮M ACC, 0.5 ␮M AVG, 10 ␮M Co2+ and 0.1 mg L−1 1-MCP. Data
are mean ± SE of four replicates and bars with different letters indicate significant
difference at P < 0.05.
3.4. Supply of organic P to P-deficient medium enhanced P
accumulation in plants

The enhanced activity of APase under P-deficient conditions

would hydrolyze organic P, thus facilitating P accumulation by
increase in APase activity was also reduced by the treatment with plants. To test this hypothesis, we measured P concentrations in
1-MCP. These results are indicative that ethylene evoked by P defi- roots and shoots of plants grown under P-deficient conditions in
ciency may play a regulatory role in modulation of APase activity.
To further test this hypothesis, we experimentally manipulated
ethylene production by treatments of roots with ethylene syn-
thesis precursor ACC. We found that ACC mimicked P deficiency
in terms of stimulation of APase under P-sufficient conditions
(Fig. 3).

3.3. Phosphorus deficiency-induced increase in expression of gene

encoding acid phosphatase (MfPAP1) is dependent on ethylene

We further identified a gene encoding secreted acid phos-

phatase in M. falcata (MfPAP1) (Xiao et al., 2006) and studies
the responses of MfPAP1 to P deficiency and ethylene levels.
As shown in Fig. 4, there was a rapid increase in MfPAP1
transcript in response to P deficiency. The P-deficiency-induced
increase in MfPAP1 transcript was markedly reduced by ethy-
lene synthesis inhibitors AVG and Co2+ . A similar increase in
MfPAP1 transcript was also observed when seedlings exposed to Fig. 4. Effect of P deficiency (−P), ACC and AVG on expression of MfPAP1 gene that
P-sufficient solution were treated with ethylene synthesis precur- is encoding a root excreted APase. M. falcata seedlings grown in P-sufficient (+P)
sor ACC (Fig. 4). These results indicate that ethylene acts as an solution were challenged with P deficiency (−P), 1 ␮M ACC, and 0.5 ␮M AVG for 6 h,
and relative mRNA level determined by qRT-PCR and was normalized based on the
important signal to mediate P-deficiency-induced up-regulation of mRNA in roots grown +P solutions. Data are mean ± SE of three replicates and bars
MfPAP1. with different letters indicate significant difference at P < 0.05.
118 Y.-S. Li et al. / Environmental and Experimental Botany 71 (2011) 114–120
Fig. 5. Changes in P concentrations in roots and shoots after 48 h exposure of M.
falcata seedlings to P-deficient (−P) solution containing 83.3 ␮M inositol phosphate
(Ino-P) in the absence and presence of 0.5 M AVG and 10 ␮M Co2+ . A similar exper-
iment was also conducted to examine the effect of Ino-P on P concentrations in
roots and shoots of P-sufficient (+P) seedlings by the addition of 83.3 ␮M inosi-
tol phosphate to P-sufficient solution (+P + Ino-P). Data are mean ± SE with four to
six replicates for each treatment and bars with different letters indicate significant
difference at P < 0.05.
the absence and presence of organic P, inositol phosphate (Ino-P).
Concentrations of P in both roots and shoots were significantly
enhanced when Ino-P were added into the P-deficient medium
(Fig. 5). The increases in P concentrations in both roots and shoots
were abolished when ethylene synthesis inhibitor AVG and Co2+
was used to treat the seedlings (Fig. 5). These results suggest that
the enhanced APase activity is involved in hydrolysis of organic
P by stimulating APase activity. In contrast to seedlings grown in
P-deficient solution, addition of the same amount of inositol phos-
phate into P-sufficient solution did not improve P status in roots
and shoots of seedlings (Fig. 5).
4. Discussion
Phytohormones such as auxin (Lopez-Bucio et al., 2002) and
ethylene (Borch et al., 1999; Ma et al., 2003; Zhang et al., 2003)
have been shown to be associated with the changes in root archi-
tecture in response to P deficiency. An evolution of ethylene from
P-deficient seedlings has been reported in a number of plant species
(Borch et al., 1999; Li et al., 2009). Our previous study demon-
strated that P-deficiency-induced ethylene played a regulatory role
in modulation of root hydraulic conductivity in M. falcata seedlings
(Li et al., 2009). In the present study, we found that the reduc-
tion in root P concentration induced by P deficiency was alleviated
by ethylene synthesis inhibitors and that treatment of P-sufficient
seedlings with ACC led to a similar reduction in root P concentra-
tion (Fig. 1). We further found that up-regulation of high-affinity
P transporters of MfPT1 and MfPT5 in response to P deficiency was
markedly inhibited by AVG, and that there was an up-regulation of
MfPT1 and MfPT5 in P-sufficient seedlings by ACC (Fig. 2). Finally,
we also demonstrated that P-deficiency-induced stimulation of
root APase activity M. falcata seedlings was significantly inhibited
by AVG and Co2+ , while ACC enhanced the root APase activity of
P-sufficient seedlings (Fig. 3). The changes in expression of gene
encoding root APase (MfPAP1) were comparable to those changes in
root APase activity in response to P deficiency and ethylene synthe-
sis inhibitors and precursor (Fig. 3), suggesting that the changes in
MfPAP1 expression underpins the observed changes in APase activ-
ity. To determine whether the enhanced root APase activity is of
physiological relevance, we measured P concentrations in roots and
shoots in the absence and presence of organic P under P-deficient
conditions. Our results showed that P concentrations in both shoots
and roots were enhanced when organic P (inositol phosphate)
was supplied to the seedlings grown in P-deficient solution. More
importantly, we found that the increases in root and shoot P con-
centrations were reversed by ethylene synthesis inhibitor (Fig. 5).
Taken together, these findings highlight the important role played
by ethylene in adaptation of plants to P deficiency by up-regulating
high-affinity phosphate transporters and acid phosphatase activity.
Unlike most studies reported in the literature (e.g.,Wasaki et al.,
2009), our study focused on the relative short-term (48 h) effect
of P deficiency and ethylene on root APase activity and P acquisi-
tion in roots and shoots. Because both P deficiency and ethylene
treatment can lead to changes in root morphology such as stimula-
tion of lateral root and root hair growth and inhibition of primary
roots (Vance et al., 2003), this would affect P uptake and root APase
activity due to changes in root surface area as the P concentra-
tion and APase activity were expressed on the basis of root weight.
The short-term effect of P deficiency and ethylene used in the
present study may minimize the potential contributions of changes
in root architecture to the observed changes in P uptake and root
APase activity. The observation that treatments with P deficiency
and ethylene for 6 h can lead to several folds increases in expres-
sion of MfPAP1 expression (Fig. 2) may argue that the changes in
APase activity by these treatments are unlikely to be accounted
for by changes in root morphology. The involvement of ethylene
in modulation of iron nutrition by regulating ferric reductases and
iron transporters in Arabidopsis under Fe-deficient conditions has
been reported by Lucena et al. (2006). The relative short-term P-
deficient treatment used in the present study may also account
for the results that no effect of P deficiency on shoot P concentra-
tion, and less effect on P concentration in roots (Fig. 1) compared to
those reported in literature (cf. Li et al., 2003). Future studies with
extended treatment period and using mutants defective in ethylene
synthesis (signaling) and/or root APase would validate the role of
ethylene played in response to P deficiency in terms of utilization
and mobilization of plant available P.
Enhanced activity of root APase has been reported in numer-
ous plant species under P-deficient conditions (see reviews of
Richardson et al., 2009; Tran et al., 2010). Our results that P-
deficiency-induced a marked increase in root APase activity in
legume plants of M. falcata are consistent with those reported in
the literature. One important finding in our study was that the P-
deficiency-induced increase in root APase activity can be mimicked
by experimentally-manipulated increases in ethylene levels using
ACC (Fig. 3). On the other hand, the enhanced APase activity evoked
by P deficiency was significantly inhibited by ethylene synthesis
inhibitors (AVG and CoCl2 ) and antagonist of ethylene action (1-
MCP) (Fig. 3). Given that there is an increase in ethylene evolution
from M. falcata roots under P-deficient conditions (Li et al., 2009);
our results demonstrate that ethylene is an important mediator to
modulate root APase activity. There has been little information on
the involvement of phytohormones in regulation of plant APase
activity in the literature. Wittenmayer and Merbach (2005) pre-
sented data showing that IAA stimulates root APase activity of
maize seedlings grown in both P-sufficient and P-deficient solu-
tions by 26% and 127%, respectively. The increase in root APase
activity in our study was much greater than IAA-induced increase
in maize root APase. For instance, ACC stimulated the root APase
activity of P-sufficient M. falcata by 187%. There is ample evidence
demonstrating the close link between ethylene and auxin in medi-
ating physiological processes in plants. For instance, a recent study
indicated that aluminum-induced inhibition of root elongation in
Arabidopsis may be mediated by an interactive effect of ethylene
and auxin with ethylene being as an up-stream signal (Sun et al.,
2010). A similar mechanism may also account for the dependence
of APase activity on auxin and ethylene.
 Y.-S. Li et al. / Environmental and Experimental Botany 71 (2011) 114–120
In addition to studying the effect of P deficiency and ethylene
on root APase activity, we also evaluated the physiological func-
tion of root APase by measuring P acquisition in the absence and
presence of substrate (inositol phosphate) for APase under vary-
ing conditions. The enhanced root APase activity under P-deficient
conditions would facilitate hydrolysis of inositol phosphate, liber-
ating available inorganic P for acquisition by plants. Our results that
supply of organic P to P-deficient seedlings significantly increased
P contents in both shoots and roots of plants exposed to P-deficient
solution, but not P-sufficient solution (Fig. 5), indicate that the
enhanced APase activity is likely to be functionally operative to
release inorganic P available for plant acquisition. Interestingly, we
found that the P concentration in shoots of M. falcata seedlings
under P-deficient conditions in the presence of organic P was higher
than that of P-sufficient seedlings (Fig. 5). We have no available
explanations for such result. But the following arguments may
account for the observation. Firstly, the enhanced root APase activ-
ity in the presence of organic P under P-deficient conditions would
facilitate the release of inorganic P from organic P for uptake by
plants. Secondly, the up-regulation of high-affinity P transporters
(Fig. 2) would maximize P uptake by plants, leading to the higher P
concentration in shoots under P-deficient conditions in the pres-
ence of inositol phosphate than in shoots of P-sufficient plants
(Fig. 5). Alternatively, plants grown in P-sufficient solution may
grow faster than those grown in P-deficient solution supplied with
organic P, resulting in the greater biomass of P-sufficient seedlings.
This would lead to a “dilution effect” such that the P concentra-
tions in P-sufficient seedlings are lower than those in P-deficient
solution supplied with organic P (Fig. 5), while the total P con-
tents in P-sufficient seedlings are likely to be higher than those
seedlings exposed to P-deficient solutions with organic P. Further-
more, we also demonstrated that the increases in P concentrations
in roots and shoots of P-deficient seedlings were reversed by ethy-
lene synthesis inhibitors (Fig. 5). Our previous studies showed that
P deficiency evoked an increase in ethylene production from M. fal-
cata roots (Li et al., 2009). Taken together, these findings prompt
us to speculate that P-deficiency-induced ethylene may act as a
signal to stimulate root APase activity, leading to liberation of inor-
ganic P for acquisition by plants. There is emerging evidence in
support of the role of ethylene played in mineral deficiency and
toxicity in the literature. These include nitrate sensing and acquisi-
tion (Leblanc et al., 2008; Tian et al., 2009), iron deficiency (Lucena
et al., 2006) and aluminum toxicity (Sun et al., 2010). Previous stud-
ies have demonstrated that ethylene plays a regulatory role in root
growth and development under P-deficient conditions (Borch et al.,
1999; Ma et al., 2003; Zhang et al., 2003). Our findings revealed that
ethylene was involved in P acquisition under P-deficient conditions
by stimulating expression of gene encoding APase (MfPAP1), lead-
ing to the observed increase in root APase activity. In addition to
up-regulation of root APase activity, both P deficiency and ethy-
lene induced up-regulation of high-affinity phosphate transporters
(cf. Fig. 2). The enhanced expression of the high-affinity phosphate
transporters would facilitate uptake of the liberated inorganic P by
root APase, thus contributing to the observed increases in root P
concentrations under P-deficient conditions. Several recent stud-
ies have reported that ethylene can regulate nitrate transporters
(Leblanc et al., 2008; Tian et al., 2009) and phosphate transporters
(Chapin and Jones, 2009). Therefore these findings highlight that
ethylene is likely to be a key component in sensing P deficiency
and modulating P mobilization and acquisition under P-deficient
conditions.
Acknowledgements
This work was supported by the State Key Basic Research Devel-
opment Program of China (2007CB106800) and Natural Science
119
Foundation of China (Nos. 30821062, 30800706 and 30788003) and
State Key Laboratory of Vegetation and Environmental Change. We
thank the two anonymous reviewers for their constructive sugges-
tions.
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