Plant Physiology Preview. Published on December 2, 2009, as DOI:10.1104/pp.109.147918
Running Title: PvPAP3 Enhancing ATP Utilization
Corresponding author:
Name: Hong LIAO
Address: Root Biology Center, South China Agricultural University, Guangzhou 510642,
China
Telephone: 86-20-85283380
Fax Numbers: 86-20-85281829
E-mail: [email protected]
Research area: Environmental Stress and Adaptation to Stress
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Copyright 2009 by the American Society of Plant Biologists
1
Biochemical and Molecular Characterization of PvPAP3, a Novel Purple Acid
Phosphatase Isolated from Common Bean Enhancing Extracellular ATP
Utilization
Cuiyue Liang1*, Jiang Tian1*, Hon-Ming Lam2, Boon Leong Lim3, Xiaolong Yan1 and
Hong Liao1
1
Ministry of Agriculture Key Laboratory of Soil and Plant Nutrition in South China, Root
Biology Center, South China Agricultural University, Guangzhou 510642, PR China
2
Department of Biology and State Key Laboratory of Agrobiotechnology, The Chinese
University of Hong Kong, Hong Kong, PR China.
3
School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong,
PR China.
* These authors contributed equally and are considered as co-first authors
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2
Running Title: PvPAP3 Enhancing ATP Utilization
Footnotes
Financial source:
This work is supported in part by grants from the National Natural Science Foundation of
China (Grant No. 30890131) and National Program on the Development of Basic
Research of China (Grant No. 2005CB120902) to X.Y. and H.L.; and the Hong Kong
RGC Earmarked Grant (CUHK4434/04M) and the Hong Kong UGC AoE Plant &
Agricultural Biotechnology Project (AoE-B-07/09) to H.-M.L.
Corresponding author:
Hong LIAO
[email protected]
Fax: 86-20-85281829
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ABSTRACT
Purple acid phosphatases (PAPs) play diverse physiological roles in plants. In this study,
we purified a novel PAP, PvPAP3 from the roots of common bean (Phaseolus vulgaris)
grown under phosphate (Pi) starvation. PvPAP3 was identified as a 34 kD monomer
acting on the specific substrate, ATP, with a broad pH range and a high heat stability. The
activity of PvPAP3 was insensitive to tartrate, indicating that PvPAP3 is a PAP-like
protein. Amino acid sequence alignment and phylogenetic analysis suggest that PvPAP3
belongs to the group of plant PAPs with low molecular mass. Transient expression of
35S:PvPAP3-GFP in onion epidermal cells verified that it might anchor on plasma
membrane and be secreted into apoplast. Pi starvation led to induction of PvPAP3
expression in both leaves and roots of common bean, and expression of PvPAP3 was
strictly dependent on P availability and duration of Pi starvation. Furthermore, induction
of PvPAP3 expression was more rapid and higher in P-efficient genotype, G19833, than
that in P-inefficient genotype, DOR364, suggesting possible roles of PvPAP3 in P
efficiency in beans. In vivo analysis using a transgenic hairy root system of common bean
showed that both growth and P uptake of bean hairy roots from the PvPAP3
overexpression transgenic lines were significantly enhanced when ATP was supplied as
the sole external P source. Taken together, our results suggest that PvPAP3 is a novel PAP
which might function in the adaptation of common bean to P deficiency, possibly through
enhancing utilization of extracellular ATP as a P source.
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INTRODUCTION
As one of the essential macronutrients, phosphorus (P) is directly or indirectly
involved in many important physiological and biochemical processes in plants
(Marschner, 1995). In soils, P is easily fixed by organic compounds, Fe or Al oxides into
the forms that are unavailable to plants. Therefore, low P availability is one of the major
factors limiting crop production (Raghothama, 1999; Vance et al., 2003). Plants have
developed a group of adaptive strategies to enhance P acquisition and utilization, such as
modifying root morphology and architecture (Liao et al., 2004; Devaiah et al., 2007a and
b; Zhou et al., 2008), increasing acid phosphatase (APase) activity (del Pozo et al., 1999;
Bozzo et al., 2002; Wang et al., 2009) and organic acid exudation (Ligaba et al., 2004), as
well as enhancing expression of a diverse array of genes (Raghothama, 1999; Vance et
al., 2003). Among them, APases are generally believed to be important for P acquisition
and utilization (Bieleski, 1973; Duff et al., 1994).
Plant APases function to hydrolyze Pi from orthophosphoric monoesters and have a
pH optimum below 7.0. They are traditionally divided into two groups according to their
substrate specificity, including non-specific versus specific APases (Duff et al., 1994).
Purple acid phosphatases (PAP) belong to a special group of APases that are
characterized by the purple color of purified proteins in water solution and tartrateinsensitive metallo-phosphatase activity (Schenk et al., 1999; Schenk et al., 2000; Li et
al., 2002). Based on their molecular mass and protein structure, PAPs can be further
divided into two groups, including small PAPs with a molecular mass of about 35 kD,
and large PAPs that are mostly homodimeric proteins with a subunit molecular mass of
about 55 kD (Schenk et al., 1999; Schenk et al., 2000; Li et al., 2002; Olczak et al.,
2003). Among all of the PAPs, seven invariant residues are highly conserved and are
required for metal coordination (Schenk et al., 1999; Schenk et al., 2000; Li et al., 2002;
Olczak et al., 2003).
Increased PAP activity by Pi starvation has been demonstrated in various plants,
including Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), potato
(Solanum tuberosum) and tomato (Lycopersicon esculentum) (del Pozo et al., 1999;
Bozzo et al., 2002; Kaida et al., 2003; Zimmermann et al., 2004), indicating that these Pi
starvation responsive PAPs, like some other APases, might be involved in P acquisition
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5
and mobilization. Transgenic expression of MtPAP1 resulted in improved P acquisition
and growth in Arabidopsis when phytate P was supplied as the sole external P source
under sterile conditions (Xiao et al., 2006). Recent results showed that overexpressing
AtPAP15 could enhance P efficiency in soybean (Wang et al., 2009). However, it has been
difficult to describe the general physiological functions of all plant PAPs due to their
diversity and ubiquity. In the Arabidopsis genome, a total of twenty nine PAP-like genes
have been identified. Among them, the transcription of only four PAP-like genes was
induced by Pi starvation, including PAP11, PAP12, PAP17 and PAP26 (Li et al., 2002;
Veljanovski et al., 2006). Furthermore, PAP17 showed both APase and peroxidase
activities, indicating a multi-functional nature for the enzyme (del Pozo et al., 1999). In
addition to P acquisition and utilization, other functions of plant PAPs, such as protection
against oxidative stress and involvement in cell wall synthesis have been suggested in
soybean (Glycine max) and tobacco (Klabunde et al., 1995; Kaida et al., 2003; Liao et al.,
2003; Li et al., 2008).
Common bean (Phaseolus vulgaris) is a major food leguminous crop, mostly
cultivated on P deficient soils in the tropics (Beebe et al., 2006). The origin of this crop
from two major gene pools representing Middle America and Andean mountain areas has
provided excellent genetic resources for characterization and improvement of bean P
efficiency (the ability to maintain yield under low P conditions). Two common bean
genotypes, G19833 (P-efficient) and DOR364 (P-inefficient), as well as their
recombinant inbred lines, have been used by several groups to elucidate the
morphological, physiological, and genetic mechanisms underlying P efficiency, including
development of a shallower root system, greater exudation of organic acids and protons,
and formation of more cortical aerenchyma in roots (Shen et al., 2002; Fan et al., 2003;
Liao et al., 2004). Genes involved in the response network of common bean to P
deficiency have been identified through the sequencing of 3,165 ESTs from a Pi-starved
root cDNA library and via in silico approaches (Ramírez et al., 2005; Graham et al.,
2006). More recently, works from our lab have suggested that enhanced expression of the
P-responsive genes, PvPT1 and PvPS2, might contribute to efficient P acquisition and
translocation in the P-efficient genotype, G19833, under P limiting conditions (Tian et al.,
2007).
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In an earlier study, one principal APase isoform in the leaves of the P-inefficient
genotype, DOR364, was found to be entirely defective in the leaves of the P-efficient
genotype, G19833, and its activity was not associated with P acquisition and utilization in
common bean (Yan et al., 2001). The study of other APases, especially the APase
isoforms induced by Pi starvation, could be more productive for understanding P
efficiency in crops such as common bean. In the present study, one Pi starvation-induced
APase isoform, PvPAP3, was purified from the roots of common bean. Subsequently its
biochemical and molecular characteristics, as well as its roles in P utilization were
demonstrated.
RESULTS
Effects of Pi starvation on APase activity
The total APase activity in common bean was significantly increased by Pi starvation
(Fig. 1A). In leaves, the APase activity generally increased by more than four folds in the
P-inefficient genotype, DOR364, but much less in the P-efficient genotype, G19833.
However, no significant difference was observed in root APase activity between G19833
and DOR364. In the non-denaturing polyacrylamide gel (PAGE) actively stained, one
APase isoform (named as PvPAP3) with the highest mobility was obviously induced by
Pi starvation in both leaves and roots of G19833 and DOR364 (Fig. 1B). Furthermore,
activity staining patterns of APase isoforms in leaves were clearly different from those in
roots. In leaves, a total of three APase isoforms were detected in DOR364 grown at low P,
including one major APase isoform which was fully defective in G19833. This might be
the reason why the total APase activity in the leaves of G19833 was much lower than that
of DOR364.
Purification and biochemical characterization of the PvPAP3 protein
PvPAP3 was purified from the roots of Pi starved G19833 using three consecutive
purification steps: selective precipitation with ammonium sulphate (60±80%), nondenaturing PAGE, and Q-sepharose ion-exchange chromatography. The purified protein
at different steps was analyzed using denaturing SDS-PAGE gels. A major protein band
with a molecular mass of approximately 34 kD was clearly and consistently detected on
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7
the gel (Fig. 2A). The molecular mass of the native PvPAP3 was estimated to be about 34
kD by gel filtration using a Sephadex G-100 column and four protein size standards (Fig.
2C). Immunoblot analysis showed that the purified PvPAP3 was recognized by a PvPAP3
antibody (Fig. 2B).
A range of pH optimum for PvPAP3 activity using ρ-NPP as substrate was found
spanning from pH 6.0 to 8.0 (Fig. 2D). To study thermal stability, PvPAP3 was incubated
at various temperatures for 15 or 30 min, and its activity against ρ-NPP was subsequently
measured. PvPAP3 activity significantly decreased as the reaction temperature was
increased from 30℃ to 70℃. PvPAP3 activity decreased by 80% at 70℃ compared with
30℃ after 30 min incubation (Fig. 2E).
The substrate specificity of purified PvPAP3 was examined (Table Ⅰ). Phosphatase
activity using ρ-NPP as substrate was set as 100% for comparison with the activity using
other substrates. Interestingly, PvPAP3 exhibited the highest activity with ATP (307%)
and the distant second activity with α-napthyl phosphate as substrates (55%). Very limited
activity was found when phytate, AMP, and GMP were used as the substrates (Table Ⅰ).
Effects of various reagents on PvPAP3 activity were also studied. PvPAP3 activity
was significantly inhibited by the application of CuSO4, NaCl, KH2PO4, CaCl2 and
ZnSO4, and stimulated by MgCl2 (Table Ⅱ). Furthermore, PvPAP3 activity was not
affected by the addition of tartrate, verifying that PvPAP3 belongs to the PAP subgroup of
APase.
Cloning, expression analysis of the PvPAP3
Using MALDI-TOF/TOF MS analysis, the following sequences of trypsin-digested
peptides of PvPAP3 were obtained: EGYTSVLGNHDYR, KPKSKLHLLVVDGWGR
and VLLVGDWGR. Comparison with sequences in databases revealed that one fragment
was highly similar to a putative PAP in Arabidopsis (AAL49808), while the other two
sequences had highest similarity to a PAP (AAP51881) in rice (Oryza sativa). The full
length cDNA coding PvPAP3 was isolated from a cDNA library constructed from
G19833 plants under P deficiency by PCR amplification using degenerate and gene
specific primers (detailed in Materials and Methods).
The PvPAP3 cDNA clone (FJ464333) contains a 993-bp open reading frame
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8
encoding a polypeptide of 331 amino acid residues. A putative signal peptide containing
29 amino acid residues was found in the N terminus, suggesting that the molecular mass
of the mature PvPAP3 protein is approximately 34 kD. Using PvPAP3 as a query
sequence with the BLASTx algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed
that the conserved domain and metal-binding residues of deduced amino acid sequence of
PvPAP3 was similar to the other PAPs from common bean, soybean, potato and
Arabidopsis. The alignment of amino acid sequences of these proteins is illustrated in
Fig. 3 with five distinct conserved motifs highlighted.
A phylogenetic tree was generated through analysis of the PvPAP3 and the protein
sequence of other PAPs including four PAPs (CAA04644, BAD05166, AAF60317 and
TC14892) in common bean (Schenk et al., 2000; Vogel et al., 2002; Yoneyama et al.,
2004). The phylogenetic analysis showed that there were two distinct PAP groups in
plants, denoted as PAPs with high molecular mass (GroupⅠ) and low molecular mass
(Group Ⅱ) (Fig. 4). Based on this analysis, PvPAP3 protein was grouped in Group
Ⅱand had the high similarity to one PAP (NP 178297) from Arabidopsis (Fig. 4).
To test whether the expression of PvPAP3 is responsive to changes in plant nutrient
status, G19833 (P-efficient genotype) plants were subjected to starvation of nitrogen (N),
potassium (K), iron (Fe), or phosphate (Pi). Transcript accumulation of PvPAP3 revealed
by qRT-PCR data was only induced in both leaves and roots under Pi starvation, but not
under the other nutrient starvation conditions (Fig. 5).
A dose response experiment was then carried out to investigate the expression
patterns of PvPAP3 in common bean in response to different P status conditions by qRTPCR. Transcript accumulation of PvPAP3 in both leaves and roots was strictly dependent
on P availability in the medium (Fig. 6A, B). In leaves and roots of G19833 and
DOR364, a noticeable accumulation of PvPAP3 transcripts was observed for plants
grown on nutrient solution containing 5, 50 and 100 µM Pi, respectively, and the
transcript abundance decreased with increasing Pi concentration. Interestingly, the
induction of PvPAP3 transcripts was stronger in both leaves and roots of G19833 than
that of DOR364 at the same P level. Furthermore, when Pi concentration in the nutrient
solution was raised from 5 to 50 µM, greatly reduced expression of PvPAP3 was only
observed in the roots of DOR364, but not of G19833.
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The time course for PvPAP3 expression in response to the onset of P deficiency was
also investigated. The induced PvPAP3 expression in the leaves and roots of both
genotypes increased with increasing duration of Pi starvation and reached highest level at
12 d after P deficiency treatment (Fig. 6C, D). However, the induction of PvPAP3 was
more rapid and intensive in G19833 compared to DOR364 in response to Pi starvation, as
indicated by that the induction of PvPAP3 in the leaves of G19833 was observed at 4 d
after Pi starvation, but at 8 d in DOR364. And the relative expression of PvPAP3 in the
leaves of G19833 was significantly greater than that of DOR364 at the same day after P
deficiency treatment. In roots, expression level of PvPAP3 was about two fold higher in
G19833 than that in DOR364 at 8 d after growing in P deficiency, but this difference was
diminished at 12 d of Pi starvation.
Subcellular localization of PvPAP3
Bioinformatics analysis showed that 29 amino acid residues in the N terminal of
PvPAP3 were predicted as signal sequences (http://www.expasy.ch/). 17 amino acids of
the
signal
peptides
were
identified
to
be
hydrophobic
(http://www.enzim.hu/hmmtop/html/submit.html). These indicated that the PvPAP3
might anchor on the plasma membrane or be secreted outside of the cells.
To determine the subcellular localization, PvPA3 was combined with GFP reporter
gene and transiently expressed in onion (Allium cepa) epidermal cell. The GFP empty
vector was used as a control. After the cells were plasmolyzed by adding 30% sucrose
solution, laser confocal scanning microscopy was used to check whether PvPAP3 was
located on plasma membrane or cell wall. The results clearly suggested that most the
signals of PvPAP3-GFP were detected on the plasma membrane and a few signals were
located in the apoplast region (Fig. 7). However, the signal of GFP of the empty vector
control was detected throughout the intracellular areas.
PvPAP3 enhances extracellular ATP utilization in transgenic bean hairy roots
Functional analysis of PvPAP3 was performed using transgenic hairy roots of
common bean. The transgenic common been hairy roots overexpressing PvPAP3 were
generated, and the overexpression of PvPAP3 was confirmed by qRT-PCR analysis using
PvPAP3 specific primers and Western blot analysis using a PvPAP3 antibody (Fig. 8A,
B). The average APase activity against ATP and ρ-NPP in 35S:PvPAP3 transgenic hairy
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10
roots were 84% and 39 % higher than those in the empty vector control, respectively (Fig.
8C, D). Furthermore, the fresh weight and P content of 35S:PvPAP3 hairy roots was
significantly higher than those of the control when 500 μM ATP was used as the sole
external P source in the growth medium, as indicated by 61% and 35% increase of the
average fresh weight and P content in 35S:PvPAP3 transgenic hairy roots, respectively
(Fig. 9).
DISCUSSION
In this study, we purified and sequenced a novel specific Pi starvation-induced APase
isoform (PvPAP3) from common bean roots. Subsequently, the full-length PvPAP3 cDNA
was isolated. Blast analysis showed that six reported ESTs in common bean had high
homology with PvPAP3 in the common bean gene index from DFCI computational
biology and functional genomics laboratory (http://compbio.dfci.harvard.edu/tgi/).
Among them, one 397-bp EST (FD795350) has 99% identities with PvPAP3, indicating
the EST is a partial fragment of PvPAP3. Expression of all the EST as related to P
availability has not been characterized except that one EST (TC7351) expression has
been reported to be up-regulated by Pi starvation in roots of common bean (Hernández et
al., 2007). However, no further information has been documented about their biochemical
and molecular properties. The biochemical properties of PvPAP3, together with its
predicted peptide sequence, strongly suggest that PvPAP3 is a bona fide PAP in common
bean. Firstly, PvPAP3 activity was resistant to tartrate (Table Ⅱ), a common property of
PAPs (Vincent and Averill, 1990; Olczak et al., 1997; Nakazato et al., 1998; Bozzo et al.,
2002), indicating that PvPAP3 belongs to the PAP subfamily of acid phosphatase.
Secondly, the predicted protein of PvPAP3 contained five motifs that are highly
conserved in other plant PAPs including seven invariant residues required for metal
coordination (Fig. 3) (Li et al., 2002). Furthermore, PvPAP3 is a monomeric protein with
a molecular mass around 34 kD determined by Sephadex G-100 chromatrography (Fig.
2C), which agrees with the molecular mass of the mature protein predicated from the
sequence of PvPAP3. This indicates that PvPAP3 belongs to the group of low molecular
mass PAPs, which are generally monomeric protein with a molecular mass of about 35
kD (Li et al., 2002). On the contrary, except the PAP recently purified from tobacco
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11
(Lung et al., 2008), most high molecular mass PAPs are homodimeric proteins with a
single subunit of about 55 kD (Schenk et al., 1999; Schenk et al., 2000; Li et al., 2002;
Olczak et al., 2003). Our further results from alignment and phylogenetic analysis of
PvPAP3 with other PAPs also revealed that PvPAP3 has a high degree of similarity with
other low molecular mass PAPs (Fig. 4). Taken together, these findings strongly suggest
that PvPAP3 is a novel PAP with low molecular mass in common bean.
Another unique feature of PvPAP3 is that it was found to be most active with ATP as
substrate, suggesting that the in vivo substrate for PvPAP3 might be ATP. It was
previously speculated that ATP is the substrate for KBPAP purified from common bean
seeds (Cashikar et al., 1997). However, despite extensive investigations made on the
understanding of biochemical properties, primary structure, x-ray structure and
subcellular localization of KBPAP, its physiological functions remained controversial
(Cashikar et al., 1997; Grote et al., 1998). In the present study, part of the PvPAP3-GFP
signals was detected in the apoplast of onion cells, while a significant fraction of the
signals was found on the plasma membrane (Fig. 7). Consistent with this results, bioinformatics analysis shows that 17 amino acids of the signal peptides on the N terminal of
PvPAP3 were predicted as hydrophobic. These results together indicate that the
hydrophobic signal peptides at the N terminus of PvPAP3 might be a transmembrane
domain and anchor the protein on the plasma membrane. However, due to the proteolytic
removal, the association of PvPAP3 with membrane might be unsteady, which could be
one of the reason for the detection of certain PvPAP3-GFP signals in the apoplast.
Another possibility is that the protein could be directly released into the extracellular
regions.
Given the high activity of PvPAP3 against ATP and the subcellular localization, we
speculate that PvPAP3 could function in P nutrition by enhancing the extracellular ATP
utilization. Our data showed that overexpressing PvPAP3 could significantly increase the
ATP hydrolyzing activity of the transgenic hairy roots (Fig. 8). Subsequently, improved
growth and P accumulation of these transgenic hairy roots were observed when ATP was
supplied as the sole P source (Fig. 9). These indicate that enhanced expression of
PvPAP3, as well as increased accumulation of PvPAP3, may help bean hairy roots to
utilize ATP in the root apoplast/rhizosphere. Consistent with our findings, Thomas et al.
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12
(1999) showed that overexpression of Apyase resulted in augmentation of the
accumulation of Pi derived from extracellular ATP in pea (Pisum sativum). Even ATP
content is generally low in soils, it has been suggested that ATP content could reach mM
levels in certain special microenvironments, such as in the regions adjacent to cell injury
or places where there is a high exocytotic activity in roots (Thomas et al., 1999; Jeter and
Roux, 2006). Thus, it is possible that utilization of ATP could be important for plant
adaptation to environments that are low in P availability, but rich in organic matter. In
addition to mobilizing phosphate from ATP, PvPAP3 might also be involved in quenching
extracellular ATP signals which can affect cytosolic Ca2+ concentration, mitogen-activted
protein kinase (MAPKs) activity, and production of NO and reactive oxygen species
(Demidchik et al., 2003; Jeter et al., 2004; Song et al., 2006; Wu et al., 2008; Wu and Wu,
2008). Since expression of PvPAP3 is also strongly induced in leaves by Pi starvation, we
can not exclude the possibility that PvPAP3 might be involved in the utilization or
signaling of extracellular ATP released by plants.
Unlike most studies regarding the expression patterns of plant PAPs in response to
environmental stresses, in this research we started by purifying the target protein and then
obtained the full-length PvPAP3 cDNA from a cDNA library of the P-efficient bean
genotype, G19833. Subsequently, the expression pattern of PvPAP3 was investigated
under different nutrient conditions. The expression of PvPAP3 was strongly induced by Pi
starvation in both leaves and roots but not by other nutrient deficiencies, including N, K
and Fe (Fig. 5), indicating that PvPAP3 was specifically regulated by P availability at the
transcriptional level. Induction of PAP expression by Pi starvation has been well
documented in other plant species such as Arabidopsis and potato (de Pozo et al., 1999;
Li et al., 2002; Zimmermann et al., 2004). It has been suggested that APase activity in
plants may contribute to improve P uptake from organic P in soils (Schachtman et al.,
1998; Rahothama, 1999; Richardson et al., 2001). Enhanced expression of PAP genes in
response to Pi starvation suggests that PAPs, like the other APases, might play an
important role in P remobilization and acquisition in plants.
It has been well documented that low P availability increases APase exudation or
enhanced internal APase activity in plants. It has also been found that there is a positive
relation between root APase activity and P uptake from inositol hexaphosphate in bean
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13
(Helal, 1990) and barley (Asmar et al., 1995), showing that APase activity might be
closely related to plant P efficiency. However, Yan et al. (2001) demonstrated that a major
APase isoform in leaves was missing in the P-efficient genotype, and thus could not
confer P efficiency in common bean. In our present study, there was no difference in total
APase activity in roots between the P-efficient genotype, G19833 and P-inefficient
genotype, DOR364, confirming that at least in common bean, overall APase activity can
not be considered a critical index for P efficiency. This could be due to the heterogeneity
of APases in plants. In the case of common bean, there are two major APase isoforms that
contribute to APase activity in roots under low P conditions. The APase studied here,
PvPAP3, is Pi starvation induced, but the other isoform is not (Fig.1), suggesting that it is
important to separate different APase isoforms in order to study their functions as related
to P efficiency. Interestingly, the speed and intensity of transcription induction of PvPAP3
were much faster and greater in the P-efficient genotype, G19833, compared to the Pinefficient genotype, DOR364 (Fig. 6), strongly suggesting the contribution of PvPAP3 to
effective P efficiency in common bean. It has been verified that the effective P efficiency
of G19833 is possibly contributed by shallower root system, exudation of more organic
acids and protons, formation of more aerenchyma in roots, as well as higher expression of
Pi transporter gene (PvPT1) (Shen et al. 2002; Fan et al. 2003; Liao et al. 2004; Tian et
al., 2007). This suggests that highly induced expression of PvPAP3, along with changes
of root morphological and physiological traits, might cause the superior P efficiency in
G19833.
In conclusion, our results demonstrate that PvPAP3 is a novel plant PAP which might
function in the adaptation of common bean to Pi starvation, possibly through its
involvement in ATP hydrolysis and the use of extracellular ATP as a P source from the
environment.
MATERIALS AND METHODS
Plant materials and growth conditions
Two common bean genotypes, G19833 and DOR364, were employed. Among them,
G19833 was characterized as a P-efficient genotype with a better adaptation to low P
conditions (Yan et al., 1995a, b).
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For protein purification, seeds of G19833 were surface sterilized for 1 min in 10%
(v/v) H2O2, then germinated in dark on germination papers soaked with 1/2 strength
modified nutrient solution as described by Yan et al. (2001). The full strength modified
nutrient solution without P was composed of (in µM) 3000 KNO3, 2000 Ca(NO3)2, 250
MgSO4, 25 MgCl2, 12.5 H3BO3, 1 MnSO4, 1 ZnSO4, 0.25 CuSO4, 0.25 (NH4)6Mo7O24, and
25 Fe-Na-EDTA. Seven days after germination, uniform seedlings were transplanted into
nutrient solution without P addition. Nutrient solution was continuously aerated and
replaced once a week. Roots were harvested for protein purification at 10 d after Pi
starvation. For analysis of APase activity and isoforms in leaves and roots, uniform
seedlings were transplanted into the nutrient solution with or without P addition (500 µM
KH2PO4). Leaves and roots were separately harvested at 10 d after treatment.
For P dose responses experiment, G19833 and DOR364 seedlings were grown for 10
d in the nutrient solution supplied with 5, 50, 100 and 250 µM KH2PO4. For time course
expression experiment, seedlings were grown in the full strength nutrient solution
supplied with 250 µM KH2PO4 for 7 d, and then were transplanted to the Pi starvation
solution. At 4, 8, 12 d after treatment, leaves and roots were separately harvested. In order
to study the response specificity to nutrients, G19833 seedlings were grown in the
nutrient solution lacking of K, N, Fe or P, respectively, and leaves and roots were
harvested 10 d after treatment. All experiments, unless otherwise mentioned, had four
biological replicates.
Purification of PvPAP3
Common bean roots were harvested and frozen at -80℃. About 80 g frozen tissues
were homogenized with a Polytron at 4℃ in 240 mL of extraction buffer containing 100
mM Tris-HC1 (pH 8.0), 0.02 mM EDTA, 25 mM ascorbic acid, 10% (v/v) glycerol, and
0.0001 % (w/v) PMSF. After filtrated through four layers of cheesecloth and centrifuged,
the supernatant was fractionated on ice with ammonium sulfate. The fraction precipitates
at 60% and 80% were collected after 2 h incubation on ice by centrifugation. The pellets
were resuspended in 20 mL of 20 mM Tris-HC1 at pH 8.0, and then centrifuged to
remove the insoluble materials. The supernatant was desalted by ultra-filtration in the
same extraction buffer.
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15
The retrieving of target protein from non-denaturing PAGE gel followed the
procedures developed by Miller et al. (2001). Briefly, proteins in the supernatant were
separated in non-denaturing 8% (w/v) polyacrylamide gels at constant voltage of 90 V for
1.5 h. A small section of the gel was cut away for activity staining as described by Yan et
al. (2001). The activity-stained section was realigned with the unstained portion of each
gel, and the gel area corresponding to the activity was excised. The gel slices were
homogenized in 20 mM Tris-HC1 at pH 8.0. After centrifuged at 12,000 g for 10 min, the
supernatant was collected and concentrated by ultra-filtration.
About 0.5 mL supernatant was loaded to Q-Sepharose FF column, which had been
connected to an ÄKTA PURIFIER system and pre-equilibrated with Tris-HCl buffer (20
mM, pH 7.5). The column was washed with Tris-HCl (20 mM, pH 7.5) buffer until the
absorbance at 280 nm decreased to a baseline. Then the column was eluted with a linear
gradient buffer (0-0.75 M, NaCl in 20 mM, Tris-HCl buffer, pH 7.5). The single activity
peak was collected and concentrated to about 250 μL using an Amicon Ultra-15.
Enzyme activity assays, protein determination and gel electrophoresis
APase activity was determined by measuring the release of nitrophenol from ρnitrophenylphosphate (ρ-NPP) in 45 mM Na-acetate buffer (pH 5.0) as described by Yan
et al. (2001). Extracts of plant tissues were incubated at 35℃ for 15 min in 2 mL of 45
mM Na-acetate buffer containing 1 mM ρ-NPP (pH 5.0). The reaction was stopped by
adding 1 mL of 1N NaOH and the absorbance was measured at 405 nm. Protein
concentration was determined by the Coomassie blue method by using bovine serum
albumin as a standard (Bradford, 1976).
The activities towards ATP and other substrates were measured according to the
methods described by Ching et al. (1987). Briefly, enzyme extracts or purified PvPAP3
were incubated with 2 mL of 20 mM Tris-HCl buffer containing 4 mM ATP or alternative
substrates at 35℃ for 15 min. The released P content was determined by the
photocolormetric method (Murphy and Riley, 1963).
To study the thermal stability of PvPAP3, the purified PvPAP3 was separately
incubated at 25, 30, 40, 50, 60 and 70℃ for 15 and 30 min, and then its activity was
measured as before. Enzyme activity at each temperature was expressed as the percentage
of the enzyme activity incubated at 25℃. SDS-PAGE was carried out as described by
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16
Cellatly et al. (1994) and stained with Coomassie blue. APase isoforms were detected as
described by Lefebvre et al. (1990). Totally, 50μg protein of extract from roots and leaves
was separated in native gel for APase isoform detection. Since protein concentration in
root extract was low, it was concentrated using ultra-filtration before being loaded into
the native gels.
Estimation of native molecular mass with Sephadex G-100 column
About 1.0 mL solution from Q-Sepharose column, concentrated with an Amicon
Ultra-15, was applied to the Sephadex G-100 (2×60 cm) column. The column was eluted
with two column volumes of Tris-HCl (10 mM, pH 8.0) buffer at the rate of 0.5 mL.min -1.
Solution in the tubes was measured at 280 nm, native molecular mass (MM) was
calculated from a plot of Ve (elution volume)/Vo (Void volume) against log MM, using
the following proteins as standards: Albumin (67.8 kD), Ovalbinium (44.6 kD),
Chymotrypsinogenb A (19.2 kD) and Ribonuclease A (14.3 kD).
Tryptic in-gel digestion
Target protein band of interest were manually excised from the SDS-PAGE gels. Gel
chips were destained with 50% (v/v) methanol cotaining 25mM ammonium bicarbonate
for 10 min twice, followed by soaking in 100% acetonitrile (ACN) until the gels turn
opaque. Vacuum-dried gel chips were rehydrated with 10 μg.mL -1 of Trypsin (Promega
Corporation, Madison, Wisconsin, USA) in 25 mM ammonium bicarbonate (pH 8.0) and
digested for 16 to 18 h at 30℃. After digestion, the liquid containing the tryptic peptides
was extracted from gel chips and transferred into a new tube.
MALDI-TOF/TOF mass spectrometry
Mass spectrometric analysis was carried out using a MALDI-TOF/TOF tandem mass
spectrometer ABI 4700 proteomics analyzer (Applied Biosystems, Foster City, USA). For
acquisition of mass spectra, 0.5 µL tryptic-digested samples were spotted onto a MALDI
plate, followed by 0.5 µL matrix solution (4 mg.mL-1 α-cyano-4- hydroxycinnamic acid
in 35 % (v/v) ACN and 1% (w/v) TFA). The MS peaks (MH+) were detected on minimum
S/N ratio ≥20 and cluster area S/N threshold ≥25 without smoothing and raw spectrum
filtering. Fragmentation of precursor ions was performed using MS-MS 1 kV positive
mode with CID on and argon as the collision gas. MS/MS spectra were accumulated from
3000 laser shots using default calibration with Glu-Fibrinopeptide B from 4700
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17
Calibration Mixture (Applied Biosystems, USA). The MS/MS peaks were detected on
minimum S/N ratio ≥3 and cluster area S/N threshold ≥15 with smoothing.
MS database searching
The MS and MS/MS data were loaded into the GPS ExplorerTM software v3.5
(Applied Biosystems, Foster City, USA) and searched against NCBInr database by
Mascot search engine version 1.9.05 (Matrix science, London, UK) using combined MS
(peptide-mass-fingerprint approach) with MS/MS (DeNovo sequencing approach)
analysis for protein identification. Known contaminant ions corresponding to keratin and
trypsin were excluded from the peak lists before database searching. Top ten hits for each
protein search were reported. Also, DeNovo ExplorerTM software v3.5 was used to deduce
the peptide sequence (sequence taq) of the selected peptide from MS scanning and the top
ten deduced peptide sequences will be submitted for protein identification using BlastP.
Isolation of full length PvPAP3 cDNA
With a pair of degenerate primers (forward: 5’-GTIRTIGGIGAYTGGGGIMG-3’ and
reverse: 5’-CYRTARTCRTGRTTICCIAG-3’), a fragment was first amplified by PCR
from the cDNA prepared from G19833 plants subjected to Pi starvation, then cloned into
pGEM-T vector (Promega Inc., USA) and sequenced. Two gene-specific primers (PR1:
5’-CAACAAATGAACTGTAGAATGC-3’ and PF1:5’-CAATCTTTTGTAGCCGATCA
G-3’) were designed according to the sequence of the fragment determined. Pairs with
PR1 and T3, PF1 and T7 primers were separately used to amplify the 5’ and 3’ terminals
of PvPAP3 from the same cDNA library. These three fragments were analyzed and
combined together by the MEGA 4.1 program to generate a full length cDNA. Multiple
sequence alignments and phylogenetic trees were constructed with Cluster X and MEGA
4.1 program, respectively.
Quantitative real-time (qRT) PCR
Total RNA was extracted using Trizol reagent according to the manual (Invitrogen
Inc., USA), and treated with DNase I. Two μg total RNA of each sample was used for the
first-strand cDNA synthesis in 20 µL reverse transcript reaction. Reverse transcription
was performed according to the protocol (Promega Inc., USA). The first-strand cDNA
was either used for SYBR Green monitored qRT-PCR (Toyobo Inc., Japan). The qRTPCR analysis was performed by Rotor-Gene 3000 (Corbett Research, Australia). The
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18
primer
pairs
were
used
for
qRT-PCR
analysis
of
PvPAP3
(5’-
GAACGCTTGGTATTCCGTGT-3’ and 5’-TGTCTTCCCCAATCTCCAAC-3’), and
housekeeping gene EF-1α (PvTC3216 from DFCI Computational Biology and
Functional Genomics Laboratory) (5’-TGAACCACCCTGGTCAGATT-3’ and 5’TCCAGCATCACCATTCTTCA-3’).
Expression of PvPAP3 in E. coli and antiserum production
The coding region of PvPAP3 was cloned into the pET28a expression vector and
transferred to E. coli (BL21) cells. To express PvPAP3, cells were grown at 37℃ till their
density reached 6.0, then 1 mM IPTG was added. Extraction and purification of PvPAP3
was conducted after 2 h incubation at 28℃ following the instruction of the His-bind
purification kit (Novagen, Germany). The purified PvPAP3 from E. coli was used to
produce PvPAP3 antibody, which was provided by GL Biochem Company (China).
Protein immunoblot analysis
Western blot was carried out as described by Miller et al. (2001) with some
modifications. Briefly, the purified PvPAP3 from plant roots and the extracted proteins
from hairy roots were resolved on SDS-PAGE, and then electrophoretically transferred to
a PVDF membrane (Bio-Rad, USA). Prior to exposure to PvPAP3 antiserum, the
membrane was incubated overnight in 0.01 M Tris buffer (pH 8.0) containing 5% (w/v)
non-fat dry milk (Bio-Rad, USA) and 0.15 M NaCl, followed by three washes with
washing buffer containing 0.01 M Tris (pH 8.0) and 0.1% (v/v) Tween 20. The blot was
then incubated 1 h with the PvPAP3 antibody (1:1000 dilution). After three washes with
the same washing buffer, alkaline-phosphatase-tagged secondary antibody was added,
and the bolt was developed.
Subcellular localization of PvPAP3-GFP fusion protein
The plasmids of the PvPAP3-GFP fusion protein constructs and 35S:GFP empty
vector were transformed into the onion (Allium cepa) epidermal cell by particle
bombardment using the helium-driven accelerator (PDS/1000, Bio-Rad). Bombardment
parameters were shown as follows: 1100 p.s.i. bombardment pressure, 1.0-mm gold
particles, a distance of 9 cm from macrocarrier to the samples, and a decompression
vacuum of 88,000 Pa. The transformed epidermal cells were cultured on MS medium for
16 h before observed by a confocal scanning microscope system (TCS SP2, Leica). For
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
19
plasmolysis, the transformed cells were treated with 30% sucrose solution. To distinguish
the cell wall, the transformed cells were stained by propidium iodide (PI).
Overexpression constructs of PvPAP3
For overexpression of PvPAP3 driven by the 35S promoter, the coding region of
PvPAP3 was amplified with primers, PvPAP3F: 5’-TCAGAAGCTTGGCATGGCGTTAT
C-3’ and PvPAP3R: 5’-CTGACGCGTGATTCTTTACCGGTCTTT-3’. The amplified
fragment was digested with HindⅢ and MluΙ and cloned into the binary vector obtained
from South China Agricultural University (Fig. S1).
Development of transgenic common bean hairy roots
Common bean hairy roots were produced as described by Cho et al. (2000) with
minor modifications. Briefly, the seeds of DOR364 were surface-sterilized by incubating
overnight in Cl2 gas produced by the mixture of 100 mL NaClO and 4.2 mL HCl.
Sterilized seeds were germinated on 1/2 MS medium in dark for 35 to 48 h and the
cotyledons were harvested. The abaxial of cotyledons were wounded with a scalpel
previously dipped into the overnight cultures of the A. rhizogenes strain (K599)
containing 35S:PvPAP3 or empty binary vector. The wounded cotyledons were incubated
with abaxial side up on filter paper pre-moistened in sterilized water for 3 d. Cotyledons
were then transferred to a solid MS medium containing 500 µg.mL -1 carbenicillin
disodium and 20 µg.mL-1 hygromycin. Hairy root tips (1 to 2 cm in length) of different
lines were separately transferred to the fresh MS medium with the same antibiotics as
mentioned above. The established hairy roots were used for assays of qRT-PCR analysis,
phosphatase activity and Western blot analysis. Four independent transgenic lines,
including two overexpressing lines and two empty controls, were separately selected and
regenerated for further analysis. In the ATP utilization experiment, 500 µM ATP was used
as the sole external P source, fresh weight of the hairy roots from control and the
overexpressor with PvPAP3 was analyzed at 7 d after growth. The hairy roots were then
kept at 75℃ until completely dried. Phosphorus content was analyzed colorimeterically
after ash digestion (Murphy and Riley, 1963). Each treatment for different lines had five
biological replicates. Data were analyzed using the SAS program (Statistical Analysis
Systems Institute, version 6.12).
ACKNOWLEDGEMENTS
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20
We are grateful to Drs. Leon Kochian from Cornell University, Yongchao Liang from
Chinese Academy of Agricultural Sciences, Lixing Yuan from China Agricultural
University, and Hong Mei from Columbia University for critical comments, Dr.
Yaoguang Liu for providing the over-expression vector and Dr. Peter M Gresshoff for
Agrobacterium rhizogenes strain K599, Ruibin Kuang and Sau-Na Tsai for technical help
and Drs. Xiurong Wang, Yingxiang Wang, Jing Zhao and Wenbing Guo for valuable
discussions.
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Figure legends
Figure 1. Activity and isoform analysis of APases in common bean grown in nutrient
solution with or without P supply for 10 days. A. APase activity in leaves and roots. Data
is the mean of four replicates with standard error; B. APase isoforms were separated by
using native PAGE with protein extracts from leaves and roots. The gel was stained for
APase activity as described by Lefebvre et al. (1990). The arrow indicates the APase
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
26
isoform corresponding to PvPAP3. F value of ANOVA was: 193.44 (P<0.001) for genotype and
75.84 (P<0.001) for P treatment in young leaves; 43.8 (P<0.001) for genotype and 25.53 (P<0.001)
for P treatment in old leaves; 0.99 (NS) for genotype and 138.83 (P<0.001) for P treatment in roots.
Figure 2. Purification, immunoblot analysis and biochemical characterization of PvPAP3.
A. SDS-PAGE analysis of purified PvPAP3 at different purification steps. Lane1: protein
marker; Lane 2: crude protein; Lane 3: protein purified with ammonium sulfate
precipitation; Lane 4: protein purified by reclaiming from the gel; Lane 5: protein
purified with Q-Sepharose FF column; B. Immunoblot analysis of purified PvPAP3; C.
Molecular size estimation of PvPAP3 using G-100 column chromatography. Ve: elution
volume, Vo: void volume, MM: molecular mass; D. pH response for the activity of
PvPAP3. Each data point represents the percentage of the maximal enzyme activity at
different pH values; E. Thermal-stability of PvPAP3. The activity was expressed as a
percentage of maximal activity at 0 min. All the data are the mean of three replicates with
standard errors.
Figure 3. Amino acid alignment of PvPAP3 with orthologous PAPs from several plant
species. AAF60316: putative purple acid phosphatase precursor in soybean (Glycine
max); NP_178298: PAP8 in Arabidopsis (Arabidopsis thaliana); AAT37529: purple acid
phosphatase in potato (Solanum tuberosom); AAF60317: putative purple acid
phosphatase precursor in common bean (Phaseolus vulgaris). The conserved sequence
motifs are boxed, and the metal-binding residues are represented by bold letters. The
putative signal peptide cleavage site of PvPAP3 is indicated by the arrow.
Figure 4. Phylogenetic tree of PvPAP3 with some selected PAP proteins in plants. The
phylogenetic tree was constructed using MEGA 4.1 programs. The Roman numerals I
and II designate the two groups of PAP proteins. Bootstrap values were indicated for
major branches as percentages. One thousand replicates were used for all possible tree
topology calculation. Except PvPAP3 (this work), the first two letters of each protein
label represent the abbreviated species name, followed by GenBank accession number or
accession number from common bean gene index. At: Arabidopsis thaliana; Gm: Glycine
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
27
max; LI: Lupinus luteus; Mt: Medicago truncatula; Nt: Nicotiana tabacum; Pv:
Phaseolus vulgaris; St: Solanum tuberosum.
Figure 5. PvPAP3 is specifically induced by Pi starvation. Different G19833 plants were
provided with nutrient solution deficient in phosphorus (-P), nitrogen (-N), potassium (K), iron (-Fe), and control solution (CK) for 10 days. Total RNA isolated from leaves and
roots was used for qRT-PCR analysis.
Figure 6. PvPAP3 expression levels as induced by Pi starvation in common bean. Dose
response in leaves (A) and roots (B). Plants were grown hydroponically for 10 days at
different Pi concentrations (5, 50, 100 and 250 µM Pi); Temporal response in leaves (C)
and roots (D). Plants were grown hydroponically under normal conditions for 7 days, and
then subjected to P deficiency for 0, 4, 8 and 12 days, respectively. Total RNA isolated
from leaves and roots of plants was used for qRT-PCR analysis. Different letters in the same
figure represent significant difference at the 0.05 level.
Figure 7. Subcellular localization of PvPAP3 fused to GFP on onion epidermal cells. The
upper two panels were the empty vector control before and after plasmolyzing. The lower
two panels were the PvPAP3-GFP construct before and after plasmolyzing. Cells were
observed by green GFP fluorescence of the PvPAP3 protein and red propidium iodide
(PI) fluorescence of the cell wall. Bars=150 µm.
Figure 8. Gene expression and protein activity analysis of transgenic hairy roots over
expressing PvPAP3. A) qRT-PCR analysis of PvPAP3 expression; B) Immunoblot
analysis of PvPAP3 protein; C) APase activity using ATP as a substrate; D) APase activity
using ρ-NPP as a substrate. CK: control hairy roots transformed with the empty vector;
OX: transgenic hairy roots over expressing PvPAP3. Data is the mean of five replicates
with standard error. Different letters represent significant difference at the 0.05 level.
Figure 9. Fresh weight and P content of hairy roots over expressing PvPAP3 (OX) and
control hairy roots (CK) grown on the medium supplied without ATP (open bar) and with
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28
500 µM ATP (solid bar) as the P source. Seven days after growth, fresh weight (A) and P
content (B) of the hairy roots were separately measured. Each bar is the mean of five
replicates with standard error. Different letters represent significant difference at the 0.05
level.
Supported information
Supplemental Figure 1. The diagram of binary construct for expression of PvPAP3 in
common bean hairy roots. P35S, the CaMV35S promoter. Nos3’, 3’ terminator from
nopaline synthase gene.
Table Ⅰ. Substrate specificity of PvPAP3
Substrate
Relative activity (%)
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29
ρ-NPP
100
ATP
307
AMP
4
GMP
5
55
α-napthyl-acid phosphate
Fructose 1,6- diphosphate
30
D-gluctose-6-phosphate
26
Phytate
4
Note: PvPAP3 activity was determined with 4.0 mM of
each substrate and expressed as the percentage of the rate
of Pi hydrolysis from ρ-NPP.
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30
Table Ⅱ. Effects of various reagents on the activity of PvPAP3a
a
Regents
Concentration (mM)
Relative activity (%)
MgCl2
5
141.43
ZnSO4
5
59.52
NaCl
5
37.14
CaCl2
5
16.19
KH2PO4
5
12.76
GuSO4
5
8.1
EDTA
5
104.76
Tartrate
5
106.67
PvPAP3 activity against ρ-NPP was determined with 5 mM of each reagent
applied in the reaction buffer and expressed as the percentage of PvPAP3
activity without application of these regents.
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31
-P
10.0
1.0
Young leaves
APase activity in leaves
(µmol.mg-1 protein. min-1)
+P
Old leaves
Roots
8.0
.08
6.0
0.6
4.0
0.4
2.0
0.2
0
APase activity in roots
(µmol.mg-1 protein. min-1)
A
0
DOR364 G19833
B
DOR364 G19833
Young leaves
Old leaves
DOR364
G19833
DOR364
-P
-P
-P
+P
DOR364 G19833
+P
+P
Roots
G19833
-P
+P
DOR364
-P +P
G19833
-P +P
Figure 1 Activity and isoform analysis of APases in common bean grown in nutrient solution with or
without P supply for 10 days. A. APase activity in leaves and roots. Data is the mean of four replicates with
standard error; B. APase isoforms were separated by using native PAGE with protein extracts from leaves
and roots. The gel was stained for APase activity as described by Lefebvre et al. (1990). The arrow indicates
the APase isoform corresponding to PvPAP3. F value of ANOVA was: 193.44 (P<0.001) for genotype and
75.84 (P<0.001) for P treatment in young leaves; 43.8 (P<0.001) for genotype and 25.53 (P<0.001) for P
treatment in old leaves; 0.99 (NS) for genotype and 138.83 (P<0.001) for P treatment in roots.
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A
B
107kD
81kD
49kD
34kD
27kD
Ve/Vo
C
1.9
PvPAP3
34kD
1.6
1.3
1.0
1.0
D
1.5
2.5
lg (MM)
2.0
%maximum activity
120
80
40
0
%maximum activity
E
3.5 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
Figure 2 Purification, immunoblot analysis and biochemical characterization of
PvPAP3. A. SDS-PAGE analysis of purified PvPAP3 at different purification
steps. Lane1: protein marker; Lane 2: crude protein; Lane 3: protein purified
with ammonium sulfate precipitation; Lane 4: protein purified by reclaiming
from the gel; Lane 5: protein purified with Q-Sepharose FF column; B.
Immunoblot analysis of purified PvPAP3; C. Molecular size estimation of
PvPAP3 using G-100 column chromatography. Ve: elution volume, Vo: void
volume, MM: molecular mass; D. pH response for the activity of PvPAP3. Each
data point represents the percentage of the maximal enzyme activity at different
pH values; E. Thermal-stability of PvPAP3. The activity was expressed as a
from on June 15, 30
2017 - min
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0 Downloaded 15
Copyright © 2009 American Society of Plant Biologists.percentage
All rights reserved.
of maximal activity at 0 min. All the data are the mean of three
replicates with standard errors.
30℃
120
80
40
0
pH
40℃
50℃
60℃
70℃
PvPAP3
NP_178298
AAF60316
AAT37529
AAF60317
-MALSSKE--RLVFRVVFVAAVLCSFITPSMAELPRFKHAPKKPQQSLHILVVGDWGRQG
MDSLRDVKPIKLIFSIFCLVIILSACN--STAELPRFVQPPE-PDGSLSFLVVGDWGRRG
MGTQRSKP--SCTIVAIFLAFCCFVS--SSKAKLESLQHAPK-ADGSLSFLVVGDWGRKG
------MA--SMKILNIFISFLLLLLFPAAMAELHRLEHPVN-TDGSISFLVVGDWGRRG
-----------MAGLGVWLAFIGVCFLN-VSALLQRLEHPVK-ADGSLSLVVIGDWGRKG
PvPAP3
NP_178298
AAF60316
AAT37529
AAF60317
TNNQSFVADQMGIVGEKLDIDFVISTGDNFYEDGLKGVDDPAFYSSFVDIYTAHSLQKTW
SYNQSQVALQMGKIGKDLNIDFLISTGDNFYDDGIISPYDSQFQDSFTNIYTATSLQKPW
AYNQSLVAFQMGVIGEKLDVDFVISTGDNFYDNGLTGVFDPSFEESFTKIYTAPSLQKKW
TFNQSQVAQQMGIIGEKLNIDFVVSTGDNFYDDGLTGVDDPAFEESFTNVYTAPSLQKNW
TYNQSEVSAQMGRVGAKLNIDFVISTGDNFYDDGLSGVDDPAFELSFSKIYTAKSLQKQW
PvPAP3
NP_178298
AAF60316
AAT37529
AAF60317
YSVLGNHDYRGDVEAQLSPALKQKDSRWLCLRSFILDGEIVEFFFVDTTPFVDEYFVDPG
YNVLGNHDYRGNVYAQLSPILRDLDCRWICLRSYVVNAEIVDIFFVDTTPFVDRYFDEPK
YNVLGNHDYRGNAKAQISHVLRYRDNRWVCFRSYTLNSENVDFFFVDTTPYVDKYFIEDK
YNVLGNHDYRGDALAQLSPILKQKDNRWICMRSYIVNTDVAEFFFVDTTPFQDMYFTTPK
YSVLGNHDYRGDVEAQLNTILQKIDPRWICQRSFIVDTEIAEFFFVDTTPFVDKYFLKPK
PvPAP3
NP_178298
AAF60316
AAT37529
AAF60317
EHTYDWEGVLPRMSYLSQLLVDVDSALAKSKAKWKMVVGHHTINSAGHHGNTEELKQILV
DHVYDWRGVLPRNKYLNSLLTDVDVALQESMAKWKIVVGHHTIKSAGHHGNTIELEKQLL
GHNYDWRGILPRKRYTSNLLKDVDLALRQSTATWKVVIGHHTIKNIGHHGDTQELLIHFL
DHTYDWRNVMPRKDYLSQVLKDLDSALRESSAKWKIVVGHHTIKSAGHHGSSEELGVHIL
DHTYDWTGVLPRDKYLSKLLKDLEIALKDSTAKWKIVVGHHPVRSIGHHGDTQELIRHLL
PvPAP3
NP_178298
AAF60316
AAT37529
AAF60317
PILEAYNVDAYINGHDHCLEHIIDKNSGIHFLTSGGGSKAWSGDVKPWSSEELQLYYDGQ
PILEANEVDLYINGHDHCLEHISSINSGIQFMTSGGGSKAWKGDVNDWNPQEMRFYYDGQ
PLLKANNVDLYMNGHDHCLEHISSLDSSVQFLTSGGGSKAWRGDTKQSEGDEMKFYYDGQ
PILQANNVDFYLNGHDHCLEHISSSDSPLQFLTSGGGSKSWRGDMNWWNPKEMKFYYDGQ
PILEANDVDMYINGHDHCLEHISSTSSQIQFLTSGGGSKAWKGDHLIKMGKMGQRFTMMD
PvPAP3
NP_178298
AAF60316
AAT37529
AAF60317
GFMSMQITESNADIIFYDVYGKPLHSWSISKDR----------GFMSVYTSEAELRVVFYDGLGHVLHRWSTLKNGVYSDI-----GFMSVHISQTQLRISFFDVFGNAIHKWNTCKFDSSDM------GFMAMQITQTQVWIQFFDIFGNILHKWSASKNLVSIM------KYLQVWRFKKSIPKLFIMIFLAKFCKLLICPRGYVMCMPYNSLI
Figure 3 Amino acid alignment of PvPAP3 with orthologous PAPs from several plant species.
AAF60316: putative purple acid phosphatase precursor in soybean (Glycine max); NP_178298:
PAP8 in Arabidopsis (Arabidopsis thaliana); AAT37529: purple acid phosphatase in potato
(Solanum tuberosom); AAF60317: putative purple acid phosphatase precursor in common bean
(Phaseolus vulgaris). The conserved sequence motifs are boxed, and the metal-binding residues are
represented by bold letters. The putative signal peptide cleavage site of PvPAP3 is indicated by the
arrow.
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MtAAX20028
54
GmAAF19820
38
PvCAA04644
54
PvTC14892
50
StAAT37527
60
AtNP 179235
42
60
LlAJ505579
GmAAN85416
49
100
PvBAD05166
AtNP 176033
94
GroupⅠ
AtNP 179405
99
100
82
AtNP 564619
AtNP 190198
AtNP 180287
AtNP 193106
GmAAK49438
100
97
50
81
30
23
AtNP 187369
NtABP96799
AtNP 178298
AtNP 172923
AtAAL47459
StAY598343
PvAAF60317
100
41
16
LlAJ617676
GroupⅡ
AtNP 566587
7
37
AtNP 178297
PvPAP3
0.2
Figure 4 Phylogenetic tree of PvPAP3 with some selected PAP proteins in plants. The phylogenetic tree was
constructed using MEGA 4.1 programs. The Roman numerals I and II designate the two groups of PAP
proteins. Bootstrap values were indicated for major branches as percentages. One thousand replicates were
used for all possible tree topology calculation. Except PvPAP3 (this work), the first two letters of each protein
label represent the abbreviated species name, followed by GenBank accession number or accession number
from common bean gene index. At: Arabidopsis thaliana; Gm: Glycine max; LI: Lupinus luteus; Mt: Medicago
truncatula; Nt: Nicotiana tabacum; Pv: Phaseolus vulgaris; St: Solanum tuberosum;.
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Relative expression
4
3
2
1
0
Relative expression
Leaves
CK
-P
-N
0.4
-K
-Fe
Roots
0.3
0.2
0.1
0
CK
-P
-N
-K
-Fe
Figure 5 PvPAP3 is specifically induced by Pi starvation.. Different G19833 plants
were provided with nutrient solution deficient in phosphorus (-P), nitrogen (-N),
potassium (-K), iron (-Fe), and control solution (CK) for 10 days. Total RNA isolated
from leaves and roots was used for qRT-PCR analysis.
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A
a
0.45
G19833
DOR364
Relative expression
Relative expression
4.5
3.0
b
b
1.5
c
c
B
a G19833
a
0.3
c
0.15
d
Relative expression
6
5
C
50 100 250 5
50 100 250 µM
a
2
b
b
c
e
0
5
4
8 12
0
4
12
d
G19833
50 100 250 µM
DOR364
a
b
0.6
b
0.3
d
8
d
e
50 100 250 5
D
DOR364
4
0
0
0.9
G19833
cd
e
Relative expression
0
DOR364
a
0
c
d
d
0
4
8 12
d
d
0
4
8
12
d
Figure 6 PvPAP3 expression levels as induced by Pi starvation in common bean. Dose response in leaves
(A) and roots (B). Plants were grown hydroponically for 10 days at different Pi concentrations (5, 50, 100
and 250 µM Pi); Temporal response in leaves (C) and roots (D). Plants were grown hydroponically under
normal conditions for 7 days, and then subjected to P deficiency for 0, 4, 8 and 12 days, respectively. Total
RNA isolated from leaves and roots of plants was used for qRT-PCR analysis. Different letters in the same
figure represent significant difference at the 0.05 level.
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GFP
PI
Bright-field
Merge
35S:GFP
35S:GFP
(plasmolyzed)
PvPAP3-GFP
PvPAP3-GFP
(plasmolyzed)
Figure 7 Subcellular localization of PvPAP3 fused to GFP on onion epidermal cells. The upper two
panels were the empty vector control before and after plasmolyzing. The lower two panels were the
PvPAP3-GFP construct before and after plasmolyzing. Cells were observed by green GFP fluorescence
of the PvPAP3 protein and red propidium iodide (PI) fluorescence of the cell wall. Bars=150 µm.
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a
a
CK1 CK2 OX1 OX2
81 kD
0.8
b
49 kD
b
0.4
34 kD
2.5
27 kD
CK1
CK2
OX1
OX2
30
a
C
a
2.0
1.5
-1
1.0
b
b
0.5
-1
0
CK1
CK2
OX1
APase activity against ρ-NPP
(µmol.mg protein. min-1)
0
APase activity against ATP
(µmol.mg protein. min-1 )
B
A
-1
Relative expression
1.2
OX2
D
a
24
18
b
c
bc
12
6
0
CK1 CK2
OX1
OX2
Figure 8 Gene expression and protein activity analysis of transgenic hairy roots over expressing PvPAP3
(OX). A) qRT-PCR analysis of PvPAP3 expression; B) Immunoblot analysis of PvPAP3 protein; C) APase
activity using ATP as a substrate; D) APase activity using ρ-NPP as a substrate. CK: control hairy roots
transformed with the empty vector; OX: transgenic hairy roots over expressing PvPAP3. Data is the mean of
five replicates with standard error. Different letters represent significant difference at the 0.05 level.
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Fresh weight (g.plate-1 )
1.6 A
a
1.2
a
0.8
0.4
P content (mg.plate-1)
c
c
c
0
0.3
b
b
CK1
CK2
c
OX1
B
OX2
a
a
b
0.2
b
c
c
c
CK1
CK2
c
0.1
0
OX1
OX2
Figure 9 Fresh weight and P content of hairy roots over expressing PvPAP3 (OX) and control
hairy roots (CK) grown on the medium supplied without ATP (open bar) and with 500 µM ATP
(solid bar) as the P source. Seven days after growth, fresh weight (A) and P content (B) of the
hairy roots were separately measured. Each bar is the mean of five replicates with standard error.
Different letters represent significant difference at the 0.05 level.
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