Journal of Experimental Botany, Vol. 66, No. 3 pp. 1017–1024, 2015
doi:10.1093/jxb/eru461 Advance Access publication 20 December, 2014
Research Paper
Pectin enhances rice (Oryza sativa) root phosphorus
remobilization
Xiao Fang Zhu1, Zhi Wei Wang1, Jiang Xue Wan1, Ying Sun1, Yun Rong Wu1, Gui Xin Li2, Ren Fang Shen3 and
Shao Jian Zheng1,*
1 State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences; Zhejiang University, Hangzhou 310058, China
College of Agronomy and Biotechnology, Zhejiang University, Hangzhou 310058, China
3 State Key Laboratory of Soil and Sustainable Agriculture, China Institute of Soil Science, Chinese Academy of Science, Nanjing
210008, China
2 * To whom correspondence should be addressed. E-mail: [email protected]
Received 12 June 2014; Revised 15 October 2014; Accepted 21 October 2014
Abstract
Plants growing in phosphorus (P)-deficient conditions can either increase their exploration of the environment (hence
increasing P uptake) or can solubilize and reutilize P from established tissue sources. However, it is currently unclear
if P stored in root cell wall can be reutilized. The present study shows that culture of the rice cultivars ‘Nipponbare’
(Nip) and ‘Kasalath’ (Kas) in P-deficient conditions results in progressive reductions in root soluble inorganic phosphate (Pi). However, Nip consistently maintains a higher level of soluble Pi and lower relative cell wall P content than
does Kas, indicating that more cell wall P is released in Nip than in Kas. P-deficient Nip has a greater pectin and
hemicellulose 1 (HC1) content than does P-deficient Kas, consistent with the significant positive relationship between
pectin and root-soluble Pi levels amongst multiple rice cultivars. These observations suggest that increased soluble
Pi might result from increased pectin content during P starvation. In vitro experiments showed that pectin releases
Pi from insoluble FePO4. Furthermore, an Arabidopsis thaliana mutant with reduced pectin levels (qua1-2), has less
root soluble Pi and is more sensitive to P deficiency than the wild type (WT) Col-0, whereas NaCl-treated WT plants
exhibit both an increased root pectin content and an elevated soluble Pi content during P-starvation. These observations indicate that pectin can facilitate the remobilization of P deposited in the cell wall. This is a previously unknown
mechanism for the reutilization of P in P-starved plants.
Key words: Phosphorus deficiency, cell wall, pectin, hemicellulose, rice, Arabidopsis.
Introduction
As an essential macronutrient, phosphorus (P) plays important roles in plant growth and development. P is a constituent
of many cellular molecules (e.g. nucleic acids, phospholipids,
ATP, etc) and is also a pivotal regulator of many biological
processes (e.g. energy transfer, protein activation, carbon and
amino acid metabolism; Marschner, 1995). Approximately
67% of the world’s potential arable land is estimated to be
P-deficient soil, and this deficiency places limitations on crop
growth and productivity (Gong et al., 2011). Furthermore,
although P is relatively abundant in many soils, the majority
of it is not available to most plants. In general, plants have
two strategies to maximize P uptake and tolerate P deficiency.
First, plants enhance P uptake by adopting morphological
changes (e.g. the production of more lateral roots and root
hairs or an increase in root biomass; Watanabe et al., 2006).
These changes enable the plant to explore increased soil volumes and thus access limited bioavailable P sources. In addition, plant roots secrete organic acids (Ae et al., 1990; Otani
et al., 1996; Zhu et al., 2012b), phosphatases (Hall, 1969;
Pammenter and Woolhouse, 1975), and other substances
© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: [email protected]
1018 | Zhu et al.
(Otani et al., 1996) that release P from insoluble P sources
in soil. Second, plants can improve the efficiency with which
they use previously assimilated P. For example, phosphohydrolases may function as a P-recycling mechanism in secondary metabolism (Gong et al., 2011). In addition, acid
phosphatase (APase) and ribonuclease (RNase) can mobilize organic P in established tissues, and this mobilized P can
then be translocated to younger, growing tissues (Yun and
Kaeppler, 2001). Nevertheless, cellular mechanisms for reutilization of the insoluble P deposited in the plant cell wall
remain relatively unexplored.
The plant cell wall is composed mainly of cellulose and of
matrix polysaccharides including pectin and hemicelluloses
(Cosgrove, 2005). Ae et al. (1996) previously reported that,
following growth in conditions of low P availability, the cell
wall polysaccharides of groundnut roots exhibit P-solubilizing
activity. Subsequently, Gessa et al. (1997) found that the
three-dimensional arrangement of the carboxyl groups in
the d-galacturonic acid component of the pectic network of
plant cell walls has a high affinity for Fe3+, and that this network may possess the ability to solubilize Fe-P and Al-P. This
finding is supported by a previous report from Nagarajah
et al. (1970), who found that polygalacturonic acid can
form a complex with Fe3+ or Al3+ through phosphate ligand
exchange, resulting in the desorption of P from clay minerals.
These previous studies therefore suggested that cell wall polysaccharides might have some involvement in the reutilization
of P sequestered within the plant cell wall. Related to this
suggestion, we recently found that P deficiency decreases the
pectin and hemicellulose 1 content of A. thaliana cell walls
(Zhu et al., 2012b). We therefore decided to determine if the
modification of cell wall components consequent upon P
deficiency could affect P remobilization within the plant.
Rice (Oryza sativa) is not only a worldwide staple crop,
but also an important monocot plant model. Using two typical rice cultivars, ‘Nipponbare’ (Nip) and ‘Kasalath’ (Kas),
which were shown to have different sensitivity to P deficiency (Gamuyao et al, 2012), this study showed a correlation between cell wall pectin and soluble inorganic phosphate
(Pi) contents. This relationship was then further verified in
other rice cultivars and by studying a pectin-deficient mutant
of A. thaliana. Our study proposes a novel mechanism for
the remobilization of P from the cell wall under P-deficient
conditions.
Materials and methods
Plant material and growth conditions
Rice (Oryza sativa) sp. japonica ‘Nipponbare’ (Nip) and indica
‘Kasalath’ (Kas) cultivars were initially used in this study, with
subsequent study of thirteen additional rice cultivars: Azucena,
Zhezhen, Taizao, Huanghuazhan, Erjiufen, Zhongsierhao,
Huhan 1B, Wuyunjing, Jiahua No.1, MH63, Widetype, 9311, and
xiushui110. Seeds were transferred to an incubator at 25 ºC for germination. Germinated seeds were then transferred to the following
treatments: +P (complete nutrient solution), –P (complete nutrient solution lacking P). The complete nutrient solution contained
114.25 mg l–1 NH4NO3, 50.375 mg l–1 NaH2PO4·2H2O, 89.25 mg l–1
K2SO4, 110.75 mg l–1 CaCl2, 405 mg l–1 MgSO4·7H2O, 1.875 mg l–1
MnCl2·4H2O, 0.0925 mg l–1 (NH4)6Mo7O24, 1.1675 mg l–1 H3BO3,
0.04375 mg l–1 ZnSO4·7H2O, 0.03875 mg l–1 CuSO4, 34.75 mg l–1
FeSO4·7H2O, 46.53 mg l–1 EDTA–Na2, solutions were renewed every
3 d. All experiments were conducted in a growth chamber with a
14-h/26 ºC day and a 10-h/23 ºC night regime, a light intensity of
400 µmol m–2 s–1 and a relative humidity of 60%. All experiments in
this study were conducted independently at least twice.
Arabidopsis thaliana (Columbia ecotype, Col-0, WT) and qua1-2
were also used. For growth on plates, following surface sterilization, seeds were germinated in Petri dishes containing agar-solidified nutrient medium. The nutrient medium consisted of the
following macronutrients: KNO3, 606.6 mg l–1; Ca(NO3)2, 944.6 mg
l–1; MgSO4, 246 mg l–1; NH4H2PO4, 11.513 mg l–1, and the following micronutrients: Fe(Ⅲ)–EDTA, 21.06 mg l–1; KCl, 3.7275 mg l–1;
H3BO3, 0.7729 mg l–1; MnSO4, 0.16902 mg l–1; CuSO4, 0.12484 mg
l–1; ZnSO4, 0.28756 mg l–1; H2MoO4, 0.017997 mg l–1; NiSO4
–1
0.026285 mg l , according to Murashige-Skoog salts (Murashige
and Skoog, 1962). The Petri dishes were placed at 4 ºC for 2 d
for vernalization, and then transferred to an environmentally
controlled growth chamber, positioned vertically as in Yang et al.
(2011), and maintained at a temperature of 24 ºC, a light intensity
of 140 µmol m–2 S–1 and a 16/8 h day/night rhythm. Growth conditions were the same for all experiments except where otherwise
specified.
For hydroponic culture, A. thaliana seedlings were first aseptically germinated on the above solid Murashige and Skoog medium.
Two weeks later, young plantlets were placed on vermiculite for an
additional 3 weeks in an environmentally controlled growth chamber. Seedlings of similar rosette diameters were then transferred to
nutrient solution containing the above mentioned Murashige-Skoog
salts for another week. The plants were subsequently subjected to
the following treatments: +P (complete nutrient solution), –P (complete nutrient solution lacking P), NaCl (complete nutrient solution
with additional 5850 mg l–1 NaCl), and –P+NaCl (complete nutrient
solution lacking P with additional 5850 mg l–1 NaCl). In –P nutrient solution, the lack of P also resulted in the lack of NH4+ as P
was applied as NH4H2PO4, thus NH4+ was compensated by the same
concentration of NH4NO3.
Effect of P deficiency on Arabidopsis growth
Seedlings (WT Col-0 and qua1-2) with a root length about 1 cm long
were transferred to Petri dishes containing agar-solidified nutrient
medium (with or without P) for 10 d, following which the photographs were taken.
Determination of soluble inorganic phosphate (Pi)
concentrations
Rice and A. thaliana roots and leaves were weighed and homogenized with a mortar and pestle in liquid nitrogen. Pi was extracted
with 4 ml of 5 % (v/v) sulphuric acid (5 M) solution. Following centrifugation at 12 000 g, 400 µl of the supernatant was transferred and
mixed with a 200 µl aliquot of 15 % (w/v) fresh ascorbic acid (pH
5.0) dissolved in ammonium molybdate. The mixture was then incubated at 37 ºC for 30 min and absorbance at 650 nm was recorded.
Pi concentration was calculated using fresh weight normalization
(Zheng et al., 2009).
Cell wall extraction and fractionation
Extraction of crude cell wall materials and subsequent fractionation
of cell wall components were performed according to Zhong and
Läuchli (1993) with minor modifications according to Yang et al.
(2011). Roots were ground up with mortar and pestle in liquid nitrogen, homogenized with 75% ethanol for 20 min in ice-cold water,
centrifuged at 8000 rpm for 10 min, following which the supernatant was removed. The pellets were then homogenized and washed
sequentially with acetone, with methanol:chloroform at a ratio of
Pectin remobilizes root phosphorus | 1019
1:1, and with methanol, each wash for 20 min each, all supernatants
being removed following centrifugation. The remaining pellets (i.e.
the cell wall materials) were dried and stored at 4 ºC for further use.
Pectin was extracted from the prepared cell wall materials (about
2 mg) by extraction three times with 1-ml hot water at 100 ºC for
1 h each. The supernatants were combined in a 5-ml tube following centrifugation at 12 000 rpm for 10 min. The residue was subsequently extracted twice with 1-ml 4% (w/v) KOH and 0.02% (w/v)
KBH4 at room temperature for 12 h. The two supernatants were then
combined in a 2-ml tube, and centrifuged at 12 000 rpm for 10 min
to obtain hemicellulose fraction 1 (HC1). Finally, the residue was
further extracted twice with 1-ml 24% (w/v) KOH and 0.02% (w/v)
KBH4 at room temperature for 12 h. The two supernatants were
combined in a 2-ml tube, and then centrifuged at 12 000 rpm for
10 min to obtain the hemicellulose fraction 2 (HC2).
Uronic acid and total polysaccharide measurement
Pectin uronic acid content was assayed according to Blumenkrantz
and Asboe-Hansen (1973), using galacturonic acid (Sigma) as
standard. Briefly, 200 µl pectin extract was incubated with 1 ml 98%
H2SO4 (containing 0.0125M Na2B4O7·10H2O) at 100 ºC for 5 min.
Following chilling, 20 µl M-hydro-dipheny (0.15%) was applied to
the solution, which was then left standing for 20 min at room temperature before the absorbance was measured spectrophotometrically at 520 nm.
Total polysaccharide contents of the hemicellulose fractions were
determined by the phenol sulfuric acid method (Dubois et al., 1956),
and expressed as glucose equivalents. Briefly, 200 µl hemicellulose 1
(HC1) extracts were incubated with 1 ml 98% H2SO4 and 10 µl 80%
phenol at room temperature for 15 min before incubation at 100 ºC
for 15 min. Following chilling, absorbance was measured spectrophotometrically at 490 nm.
P retention in cell wall materials
As plant P status changes with respect to growth phases, and
to reflect relative differences between the +P and –P treatments,
we calculated the percentage of P contained in the cell wall in
the –P treatment to that in the +P treatment. To determine the
P retention in cell walls, 5 mg cell wall materials were placed in a
2-ml Eppendorf tube filled with 1 ml 2 N HCl, following which
the solution was shaken on a rotary shaker for 3 d. The samples
were then centrifuged at 23 000g and the supernatant collected
for P concentration determination by inductively coupled plasma
atomic emission spectroscopy (ICP-AES; Fisons ARL Accuris,
Ecublens, Switzerland).
Root exudate collection and pH measurement
For organic acid and pH analysis, Nip and Kas were first grown in
complete nutrient solution for 4 weeks, following which seedlings
were transferred to +P or –P nutrient solution for a further week. The
solutions were renewed every 3 d, whilst the pH was measured daily
with a pH electrode (Sartorius, Gottingen, Germany). Following 6 d
of culture, roots were exposed to 55.5 mg l–1 CaCl2 solution (pH 5.6)
for 24 h to collect root exudates. Roots were harvested, then washed
with deionized water, and their fresh weights were recorded.
Measurement of organic acid efflux
Root exudates (as above) were first passed through a cation exchange
column (16 mm×14 cm) filled with 5 g Amerlite IR-120B resin (H+
form, Muromachi chemical, Tokyo, Japan), and then through an
anion exchange column filled with 1.5 g Dowex 1 × 8 resin (100–200
mesh, formate form). Organic acid anions retained in the anion
exchange resin were eluted with 15 ml 1 M HCl, and the eluent was
concentrated to dryness using a rotary evaporator at 40 ºC. The
residue was re-dissolved in 1 ml 1200.3 mg l–1 NaOH and filtered
(0.2 µm) before analysis. Organic acid anions were detected by ion
chromatography (ICS 3000; Dionex) equipped with an IonPac AS11
anion-exchange analytical column (4 × 250 mm) and a guard column
(4 × 50 mm). The mobile phase was 1200 mg l–1 NaOH at a flow rate
of 0.6 ml min–1.
Statistical analysis
Each treatment had at least three replicates, each experiment was
repeated at least twice, and one set of representative data are shown
in the results. Data were analysed by one-way ANOVA and the
means were compared by Student’s t test. Different letters on the
histograms indicate that the means were statistically different at
P<0.05 level.
Results
To investigate P reutilization in rice, Nip and Kas plants were
cultivated in phosphate-deficient (–P) nutrient solutions, then
root and shoot soluble inorganic phosphate (Pi) content were
measured once a week. Whilst root and shoot soluble Pi content decreased over time of exposure to P deficiency in both
varieties, Nip consistently maintained higher root soluble Pi
levels than did Kas (Fig. 1A), whereas this increment is not
Fig. 1. Soluble inorganic phosphate (Pi) content in roots (A) and shoots (B) of rice cultivars Nip and Kas. Seedlings were exposed to P-deficient nutrient
solutions for 5 weeks (see Materials and methods). Soluble Pi content was measured every 7 d. Data are means±SD (n=4). Asterisks indicate a significant
difference at P<0.05 by Student’s t test.
1020 | Zhu et al.
significant in the shoot except at day 7 (Fig. 1B). Therefore,
in the following experiments, we mainly focus on the change
of root P.
Root cell walls were extracted and the P retained in the cell
walls was measured. Nearly 50% of total P was present in the
cell walls of both rice cultivars. As P content is changeable in
different stage of plant growth, we calculated the percentage
of P contained in the cell wall in the –P treatment to that in the
+P treatment at the same treatment time to make it comparable between different cultivars. Nip always exhibited a lower
–P/+P percentage than did Kas as indicated by the dynamics
of change in cell wall P content over time of exposure to P
deficiency, especially within the 3 weeks of P-deficient treatment (Fig. 2). As on the start day of the treatment the percentage is 100 for both cultivars, it means that much more
P was released from the cell wall of Nip on the first 3 weeks
than that from Kas. As the largest difference between Kas
and Nip was observed on the 7th day of treatment, all subsequent experiments were performed at this time point.
P deficiency was associated with a decreased pectin content in Kas, whereas Nip pectin content was unaffected by
P starvation (Fig. 3A; uronic acid levels provide a measurement of pectin content). Thus, the root cell walls of P-starved
Nip plants have a greater pectin content than the roots of
P-starved Kas plants (Fig. 3A). In contrast, whereas the
hemicellulose 1 (HC1) and hemicellulose 2 (HC2) uronic
acid contents of both Nip and Kas were relatively unaffected
by P starvation (Fig. 3B, C), the HC1 content indicated by
total sugar content decreased during P starvation in Kas but
increased in Nip, whereas the HC2 content was relatively
unaffected (Fig. 4).
The above described changes in soluble Pi level and cell
wall polysaccharide content upon P starvation are suggestive
of a general relationship between the two. To test this possibility, an array of 15 different rice cultivars were used (Fig. 5),
and a general positive correlation between root-soluble Pi and
pectin contents was found (this correlation was significant if
the cultivar Huanghuazhan was excluded from the regression
analysis; Fig. 5A). In contrast, no relationship was observed
with HC1 content (Fig. 5B), suggesting that root-soluble Pi
level is related to cell wall pectin content.
It has previously been reported that the secretion of organic
acids may affect the capacity for cell wall binding of cations
Fig. 2. Retention of P in the cell walls of roots of P-starved Kas and Nip
plants. Seedlings were exposed to P-sufficient or P-deficient nutrient
solutions for 5 weeks. P retained in the cell wall was determined from
the percentage of the cell wall P content of plants grown in P-starvation
conditions (–P) versus the cell wall P content of plants grown in P sufficient
conditions (+P). Data are means±SD (n=4). Asterisks indicate a significant
difference at P<0.05 by Student’s t test.
Fig. 4. Cell wall polysaccharide contents of P-starved and control plants
(as determined by sugar content analysis). Cell wall polysaccharides were
fractionated into HC1 and HC2 components for subsequent total sugar
content analysis. Seedlings were exposed to P-sufficient or P-deficient
nutrient solutions for 7 d, and total sugar content was measured. Data are
means±SD (n=4). Columns with different letters are significantly different at
P<0.05.
Fig. 3. Cell wall polysaccharide contents of P-starved and control plants (as determined by uronic acid content analysis). Cell wall polysaccharides were
fractionated into pectin, HC1, and HC2 components for subsequent uronic acid content analysis. Seedlings were exposed to P-sufficient or P-deficient
nutrient solutions for 7 d, and uronic acid content was measured. Data are means±SD (n=4). Columns with different letters are significantly different at P<0.05.
Pectin remobilizes root phosphorus | 1021
such as Al (Li et al., 2009). However, there was no significant
differences in either pH value (dependent on proton extrusion) or organic acid (malate, citrate, and oxalate) secretion
between Nip and Kas (irrespective of P status; Fig. 6), suggesting that organic acid efflux and acidification are unlikely
to promote root P mobilization during P starvation.
We conducted in vitro experiments to test whether pectin
can release Pi from an insoluble P source and found that
pectin can release Pi from FePO4 in vitro (Fig. 7). To verify
the relationship between pectin and soluble Pi levels, and to
determine if this effect is more generally characteristic of
plant roots (i.e. rather than being a specific characteristic
of rice roots), we next tested the relationship in the dicots
model plant Arabidopsis thaliana. The qua1-2 mutant has a
reduced cell wall pectin content (Bouton et al., 2002), and it
has altered responses to the P status of the growth medium.
In P-deficient conditions, the roots of qua1-2 mutants were
shorter than those of WT (Col-0) controls, whereas in control P conditions qua1-2 mutant roots were longer than those
of WT controls (Fig. 8A). Consistent with these findings,
in P-deficient conditions, the soluble Pi content of qua1-2
mutant roots was less than that of WT although the soluble
Pi content in the qua1-2 mutant was much higher than that
of WT in the +P conditions, which might be due to the lower
fixation of Pi in the cell wall in the mutant under P-sufficient
condition (Fig. 8B). Furthermore, as it was reported that
NaCl treatment can increase pectin content in the cell wall
(Schmohl and Horst, 2000), we treated the Arabidopsis plants
with 100 mM 5850 mg l–1 NaCl too, and found that the NaCltreated Col-0 can elevate the soluble Pi content concomitant
with the increment of the pectin content under P-deficient
conditions (Fig. 9), further demonstrating the role of pectin
in the remobilization of P in cell wall.
Discussion
It has been known for some time that plants remobilize previously assimilated P as a strategy for coping with P starvation
(Raghothama and Karthikeyan, 2005). For example, when
plants are suffering from P deficiency, organic and inorganic
P in existing cells, tissues, and organs is decomposed and solubilized and thus released for reutilization in newly growing
tissues of the plant. Accordingly, older leaves tend to senesce
more rapidly during P starvation, because their reutilized P
Fig. 5. Identification of a correlation between root soluble inorganic phosphate (Pi) and cell wall pectin contents. (A) Root soluble Pi content correlates
with uronic acid determined pectin content. (B) Root soluble Pi content does not correlate with HC1 content. Fifteen different rice cultivars were analysed
(HuangHuaZhan was treated as an outlier in A). Data are means±SD (n=4).
Fig. 6. Proton extrusion (A) and organic acid secretion (B) are shown for four-week-old seedlings grown in the presence or absence of P. Data are
means±SD (n=4).
1022 | Zhu et al.
is translocated to the newly growing tissues. In contrast, root
integrity needs to be maintained to allow systematic uptake
and transport of water and nutrients to the shoots. It is therefore potentially important for plants to maximize the utilization of P stored in the non-metabolically active parts of
the roots (such as the cell wall) in conditions where soil P
availability cannot meet the requirements for root growth.
Here, we have determined that root cell wall P is a source of
reutilized P during P starvation, and that pectin plays a role
in releasing P from that source. As expected, about half of
the P was present in the matrix space in the cell walls of rice
root (Oryza sativa), thus indicating that cell wall P is indeed a
potential source of P remobilization during P starvation. All
Fig. 7. Pectin solubilizes P from FePO4 in vitro. 50 mg FePO4 in 4 ml H2O
was treated with or without pectin for 3 d. Solubilized P was calculated
from the soluble inorganic phosphate (Pi) content in the supernatant.
Data are means±SD (n=4). Columns with different letters are significantly
different at P<0.05.
these results demonstrated for the first time that P deposited
in the cell wall can be reutilized and that cell wall pectin contributes to P remobilization. The conclusions are based on
the following observations.
First, root soluble Pi content correlates positively with root
pectin content in Nip, Kas, and a further group of 15 assayed
rice cultivars. Before the P reutilization, stored P needs to be
resolubilized, and this is why we initially measured changes
in root soluble Pi during the course of a 5-week period of P
starvation in Nip and Kas, showing that whilst the soluble Pi
level in the roots of both varieties decreased with time, the
soluble Pi content of Nip was always higher than that of Kas
(Fig. 1A), whereas the relative amount of P retained in the
cell wall of –P roots (versus +P roots) was always higher in
Kas (Fig. 2). These initial observations suggested that the P
deposited in the cell wall of Nip is more easily solubilized
and hence contributes to the higher soluble Pi of Nip. As
plant cell wall is composed of a matrix of polysaccharides,
with cellulose, hemicellulose, and pectin being major components, the levels of pectin and hemicelluloses were examined.
Although there was no difference between Nip and Kas at
the start of the treatment, Nip had significantly higher pectin
and HC1 contents after one week of –P treatment (Figs 3,
4). These observations suggested that the higher pectin and
HC1 contents of Nip might be related to the higher soluble
Pi content of Nip. However, although HC1 content correlates
well with Al accumulation in A. thaliana, rice, and triticale
(Liu et al., 2008; Yang et al, 2008; Zhu et al., 2012a), and
Cd retention in rice and A. thaliana (Xiong et al, 2009; Zhu
et al; 2012b), there was no significant correlation between
HC1 and root soluble Pi contents among the 15 assayed rice
cultivars (Fig. 5). However, a strong positive correlation was
Fig. 8. The phenotype of WT (Col-0) and the qua1-2 mutant. A. thaliana seedlings grown in P-deficient (–P) or P-sufficient (+P) conditions (A), soluble
inorganic phosphate (Pi) content of roots (B), and shoots (C). Seedlings about 1 cm in length were grown on P-deficient or P-sufficient media for a further
10 d. Data are means±SD (n=4). Columns with different letters are significantly different at P<0.05. (This figure is available in colour at JXB online.)
Pectin remobilizes root phosphorus | 1023
Fig. 9. Root soluble inorganic phosphate (Pi) content (A), shoot soluble Pi content (B), and pectin uronic acid content (C) of WT (Col-0). A. thaliana
seedlings exposed to P-deficient nutrient solution for 1 week in the presence and absence of 5850 mg l–1 NaCl. Data are means±SD (n=4). Columns with
different letters are significantly different at P<0.05.
observed between the pectin and soluble Pi contents of these
rice cultivars (Fig. 5), thus further confirming the relationship
between pectin and soluble Pi content in roots of P-starved
plants. However, Zhu et al. (2012b) previously reported a significant decrease of pectin content in P-starved A. thaliana,
which is consistent with Kas, and this may be due to different
effects of P deficiency on pectin content in different plant species or cultivars within the same species.
Second, pectin can release Pi from insoluble P sources.
Pectin has previously been shown to be a cation adsorber.
The negatively charged carboxylic groups of pectin have a
particularly high affinity for Al3+, Fe3+, and Cd2+ (Blamey
et al., 1990; Chang et al., 1999; Zhu et al., 2012b), an affinity which even exceeds the capacity to bind Ca2+ (for review,
see Rengel and Zhang, 2003). PO43– also has high affinity for
Fe3+, Al3+, and Ca2+, resulting in the formation of insoluble FePO4, AlPO4, and Ca3(PO4)2. For example, AlPO4 is
thought to be deposited in the apoplast of buckwheat when
there is Al3+ in the growth medium (Zheng et al., 2005).
Moreover, the carboxylate group (–COO–) in pectin can chelate Fe3+, and PO43– can be further trapped with a linkage of
–COO–Fe–PO4. Increasing –COO– will bind Fe more tightly
to the pectin and facilitate the release of pectin-trapped
PO43–. We tested this hypothesis by incubating insoluble
FePO4 with pectin, and found that a significant amount of
Pi was released owing to the chelation of pectin –COO– with
Fe3+ (Fig. 7). It is therefore reasonable to propose that the
higher the pectin content of the cell wall of a rice cultivar,
the greater the ability of that cultivar to solubilize insoluble
P from the cell wall will be; in other words, the change of the
pectin content in cell wall seems to be an adaptive response
to P deficiency. This is further supported by our observed
correlation between pectin and soluble Pi contents amongst
different rice cultivars (Fig 5).
Third, pectin content is positively related to soluble Pi content and P sensitivity in A. thaliana. Zhu et al (2012b) previously showed that P deficiency decreases the pectin content
of A. thaliana, Here, the pectin-reduced mutant qua1-2 displayed both increased sensitivity to P deficiency and a significantly reduced root soluble Pi content under P-deficient
conditions (Fig. 8). In addition, as reported by Schmohl and
Horst (2000), treatment of maize suspension cells with NaCl
causes an increase in cell wall pectin content, and consequent
increase in Al bound to the cell wall, and in cell sensitivity
to aluminium stress. Therefore, after A. thaliana was treated
with NaCl, both pectin and soluble Pi contents were significantly increased (Fig. 9), further confirming the positive relationship between pectin content and soluble Pi in the roots.
Finally, secretion of neither protons nor organic acids
from rice roots was detected under –P conditions. It has previously been reported that P deficiency can induce the extrusion of protons and the secretion of organic acids (Johnson
et al., 1996; Neumann et al., 1999; Dinkelaker et al., 1989;
Zhu et al., 2012b). Organic acids such as citric and piscidic
acids can form complexes with Fe3+, resulting in the release
of Pi from minerals (Gardner, 1983; Ae et al., 1990 1993).
The present study rules out the involvement of proton extrusion or organic acid secretion in the cell wall P remobilization
mechanism.
Intriguingly, Kas has actually been reported to be more
(not less) tolerant of P deficiency than Nip, because it contains a Kas-specific gene, PHOSPHORUS STARVATION
TOLERANCE 1 (PSTOL1) (Gamuyao et al., 2012).
OsPSTOL1 is expressed in root primordia and encodes a Ser/
Thr protein kinase that acts as an enhancer of root growth.
Overexpression of PSTOL1 can enhance root growth and
thus increases rice grain yield in P-deficient soils. Kas probably has a more vigorous root system when soil P availability is limited, and this increased root system vigour probably
increases P uptake because it increases the volume of soil
explored. However, when a rice plant enters the reproduction stage, it will transport most of the photosynthates to the
grains, and much less is available for root growth to explore
more P sources in soils. This present study has defined an
additional mechanism for tolerance of P deficiency, wherein
pectin enhances the mobilization of P deposited in the root
cell wall, a mechanism which is more powerful in Nip than
in Kas. We propose that these two mechanisms are complementary routes to the achievement of the same final goal,
the exploitation of both external and internal resources to
meet the plant demand for P under conditions of P-supply
limitation.
1024 | Zhu et al.
In conclusion, the present study for the first time demonstrates that cell wall pectin enhances P remobilization in rice
grown in conditions where P availability is limited. In those
varieties where root cell wall pectin content is relative higher
under low available P, the higher pectin content helps to solubilize P fixed in the cell wall, thus making it available for
reutilization.
Acknowledgements
This research was supported by the “973” programme (2014CB441002),
Natural Science Foundation of China (31372127), Program for Innovative
Research Team in Universities (IRT1185), and the Fundamental Research
Funds for the Central Universities. Thanks are given to Nicholas P. Harberd
from Oxford University for his critical reading and editing of the text and
the three anonymous reviewers for their valuable comments to improve the
quality of our work.
References
Ae N, Arihara J, Okada K, Yoshihara T, Johansen C. 1990.
Phosphorus uptake by pigeonpea and its role in cropping systems of
Indian subcontinent. Science 248, 477–480.
Ae N, Arihara J, Okada K, Yoshihara J, Otani T, Johansen C. 1993.
The role of piscidic acid secreted by pigeonpea roots grown in an Alfisol
with low-P fertility. In: Randall PJ, ed. Genetic aspect of plant mineral
nutrition. Dordrecht: Kluwer Academic Publishers, 279–288.
Ae N, Otani T, Makino T, Tazawa J. 1996. Role of cell wall of groundnut
roots in solubilizing sparingly soluble phosphorus in soil. Plant and Soil
186, 197–204.
Blamey FPC, Edmeades DC, Wheeler DM. 1990. Role of cationexchange capacity in differential aluminum tolerance of Lotus species.
Journal of Plant Nutrition 13, 729–744.
Blumenkrantz N, Asboe-Hansen G. 1973. New method for quantitative
determination of uronic acids. Analytical Biochemistry 54, 484–489.
Bouton S, Leboeuf E, Mouille G, Leydecker MT, Talbotec J, Granier
F, Lahaye M, Höfte H, Truonga HN. 2002. QUASIMODO1 encodes a
putative membrane-bound glycosyltransferase required for normal pectin
synthesis and cell adhesion in Arabidopsis. The Plant Cell 14, 2577–2590.
Chang YC, Yamamoto Y, Matsumoto H. 1999. Accumulation of
aluminium in the cell wall pectin in cultured tobacco (Nicotiana tabacum
L.) cells treated with a combination of aluminium and iron. Plant, Cell and
Environment 22, 1009–1017.
Cosgrove DJ. 2005. Growth of the plant cell wall. Nature Reviews
Molecular Cell Biology 6, 850–861.
Dinkelaker, Rӧmheld B, Marschner VH.1989. Citric acid excretion and
precipitation of calcium in the rhizosphere of white lupin (Lupinus albus L.).
Plant, Cell and Environment 12, 285–292.
Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. 1956.
Colorimetric method for determination of sugars and related substances.
Analytical Chemistry 28, 350–356.
Gamuyao R, Chin JH, Pariasca TJ, Pesaresi P, Catausan S, Dalid
C, Slamet -LI, Tecson- MEM, Wissuwa M, Heuer S. 2012. The
protein kinase Pstol1 from traditional rice confers tolerance of phosphorus
deficiency. Nature 488, 535–539.
Gardner WK, Barber DA, Parbery DG. 1983 The acquisition of
phosphorus by Lupinus albus L. III, The probable mechanism by which
phosphorus movement in the soil/root interface is enhanced. Plant and
Soil 70, 107–124.
Gessa C, Deiana S, Premoli A, Ciurli A. 1997. Redox activity of caffeic
acid towards iron (III) complexed in a polygalacturonate network. Plant and
Soil 190, 289–299.
Gong YM, Guo ZH, He LY, Li JS. 2011. Identification of maize genotypes
with high tolerance or sensitivity to phosphorus deficiency. Journal of Plant
Nutrition 34, 1290–1302.
Hall JL. 1969. Histochemical localization of β-glycerophosphatase activity
in young root tips. Annals of Botany 33, 399–406.
Johnson JF, Allan DL, Vance CP, Weiblen G. 1996. Root carbon
dioxide fixation by phosphorus deficient Lupinus albus. Contribution to
organic acid exudation by proteoid roots. Plant Physiology 112, 19–30.
Liu Q, Yang JL, He LS, Li YY, Zheng SJ. 2008. Effect of aluminum on
cell wall, plasma membrane, antioxidants and root elongation in triticale.
Biologia Plantarum 52, 87–92.
Li YY, Yang JL, Zhou Y, Zheng SJ. 2009. Protecting cell walls from
binding aluminum by organic acids contributes to Al resistance. Journal of
Integrative Plant Biology 51, 574–580.
Marschner H. 1995. Mineral nutrition of higher plants. San Diego:
Academic Press.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and
bioassays with tobacco tissue culture. Physiological Plantarum 15,
473–496.
Nagarajah S, Posner AM, Quirk JP. 1970. Competitive adsorption of
phosphate with polygalacturonate and other organic anions on kaolinate
and oxide surface. Nature 228, 83–84
Neumann G, Massonneau A, Martinoia E, Rӧmheld V. 1999.
Physiological adaptations to phosphorus deficiency during proteoid root
development in white lupin. Planta 208, 373–382.
Otani T, Ae N, Tanaka H. 1996. (P) uptake mechanisms of crop grown in
soils with low P status. II. Significance of organic acids in root exudates of
pigeonpea. Soil Science and Plant Nutrition 42, 533–560.
Pammenter NW, Woolhouse HW. 1975. The utilization of P-N
compounds by plants. II. The role of extracellular root phosphatases.
Annals of Botany 39, 347–61.
Raghothama KG, Karthikeyan AS. 2005. Phosphate acquisition. Plant
Soil 274, 37–49.
Rengel Z, Zhang WH. 2003. Role of dynamics of intracellular calcium in
aluminium toxicity syndrome. New Phytologist 159, 295–314
Schmohl N, Horst WJ. 2000. Cell wall pectin content modulates
aluminum sensitivity of Zea mays (L.) cells grown in suspension culture.
Plant, Cell and Environment 23, 735–742
Watanabe T, Osaki M, Yano H, Rao IM. 2006. Internal mechanisms of
plant adaptation to aluminum toxicity and phosphorus starvation in three
tropical forages. Journal of Plant Nutrition 29, 1243–1255.
Xiong J, An LY, Lu H, Zhu C. 2009. Exogenous nitric oxide enhances
cadmium tolerance of rice by increasing pectin and hemicellulose contents
in root cell wall. Planta 230, 755–765.
Yang JL, Li YY, Zhang YJ, Zhang SS, Wu YR, Wu P, Zheng SJ. 2008.
Cell wall polysaccharides are specifically involved in the exclusion of
aluminum from the rice root apex. Plant Physiology 146, 602–611.
Yang JL, Zhu XF, Peng YX, Zheng C, Li GX, Liu Y, Zheng SJ. 2011.
Cell wall hemicellulose contributes significantly to Al adsorption and root
growth in Arabidopsis. Plant Physiology 155, 1885–1892.
Yun SJ, Kaeppler SM. 2001. Induction of maize acid phosphatase
activities under phosphorus starvation. Plant and Soil 237, 109–115.
Zheng LQ, Huang FL, Narsai R, Wu JJ, Giraud E, He F, Cheng
LJ, Wang F, Wu P, Whelan J, Shou HX. 2009. Physiological and
transcriptome analysis of iron and phosphorus interaction in rice seedlings.
Plant Physiology 151, 262–274.
Zheng SJ, Yang JL, He YF, Yu XH, Zhang L, You JF, Shen RF,
Matsumoto H. 2005. Immobilization of aluminum with phosphorous
in roots is associated with high Al resistance in buckwheat (Fagopyrum
esculentum Moench). Plant Physiology 138, 297–303.
Zhong H, Lauchli A. 1993. Changes of cell wall component and polymer
size in primary roots of cotton seedlings under high salinity. Journal of
Experimental Botany 44, 773–778.
Zhu XF, Shi YZ, Lei GJ et al. 2012a. XTH31, encoding an in vitro XEH/
XET-active enzyme, regulates aluminum sensitivity by modulating in vivo
XET action, cell wall xyloglucan content, and aluminum binding capacity in
Arabidopsis. The Plant Cell 24, 4731–4747.
Zhu XF, Lei GJ, Jiang T, Liu Y, Li GX, Zheng SJ. 2012b. Cell wall
polysaccharides are involved in P-deficiency-induced Cd exclusion in
Arabidopsis thaliana. Planta 236, 989–997.