Biochimica et Biophysica Acta 1864 (2016) 427–434

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Biochimica et Biophysica Acta

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

Identification of phosphorus deficiency responsive proteins in a high

phosphorus acquisition soybean (Glycine max) cultivar through
proteomic analysis
Aihua Sha a,⁎, Ming Li b, Pingfang Yang b,c,⁎
a
Hubei Collaborative Innovation Center for Grain Industry, Yangtze University, Jingzhou 434023, China
b
Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, the Chinese Academy of Sciences, Wuhan 430074, China
c
Sino-African Joint Research Center, Chinese Academy of Sciences, Wuhan 430074, China

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

Article history: As one of the major oil crops, soybean might be seriously affected by phosphorus deficiency on both yield and
Received 26 October 2015 quality. Understanding the molecular basis of phosphorus uptake and utilization in soybean may help to develop
Received in revised form 7 January 2016 phosphorus (P) efficient cultivars. On this purpose, we conducted a comparative proteomic analysis on a high P
Accepted 3 February 2016
acquisition soybean cultivar BX10 under low and high P conditions. A total of 61 unique proteins were identified
Available online 4 February 2016
as putative P deficiency responsive proteins. These proteins were involved in carbohydrate metabolism, protein
Keywords:
biosynthesis/processing, energy metabolism, cellular processes, environmental defense/interaction, nucleotide
Soybean metabolism, signal transduction, secondary metabolism and other metabolism related processes. Several pro-
Phosphorus efficiency teins involved in energy metabolism, cellular processes, and protein biosynthesis and processing were found
Phosphorus deficiency to be up-regulated in both shoots and roots, whereas, proteins involved in carbohydrate metabolism appeared
Proteomic analysis to be down-regulated. These proteins are potential candidates for improving P acquisition. These findings provide
a useful starting point for further research that will provide a more comprehensive understanding of molecular
mechanisms through which soybeans adapt to P deficiency condition.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Phosphorus (P) is an essential macronutrient for plant growth and
development [1,2]. As phosphorus is often deficient from soil or exists
in unavailable forms that cannot be directly utilized by plants [3,4],
crops require a large amount of P fertilizer to maintain normal growth
in more than 30% of the world's arable land [5]. The application of P fer-
tilizer improves crop production, but at the expense of causing severe
environmental pollution and depletion of non-renewable P rocks [1,5].
Therefore, it is necessary to develop environmentally–friendly and eco-
nomically feasible strategies to improve crop production in soil with
low P. One effective solution is to develop P-efficient cultivars based
on the molecular understanding of P uptake and utilization in plants.
Abbreviations: 2D, two-dimensional; ACN, acetonitrile; APase, acid phosphatase; DTT,
dithiothreitol; GmEXPB2, Glycine max β-expansins; GRF, general regulatory factor; IEF, iso-
electric focusing; IPG, immobilized pH-gradient; EST, expressed sequence tag; MALDI-
TOF/TOF, matrix-assisted laser desorption/ionization time-of-flight-time-of-flight; MS,
mass spectrometry; NADPH, nicotinamide adenine dinucleotide phosphate; P, phospho-
rus; PAP, purple acid phosphatase; qRT-PCR, quantitative real time-polymerase chain re-
action; RAD23, radiation sensitive23; RuBisCO, ribulose bisphosphate carboxylase; SDS-
PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
⁎ Corresponding author.
E-mail addresses: aihuasha@163.com (A. Sha), yangpf@wbgcas.cn (P. Yang).
Soybean (Glycine max (L.) Merr) is one of the most widely grown
leguminous crops in the world, providing nutrition and oil for both
human diet and animal husbandry [6]. However, the production and
quality of soybean are seriously affected by the low P availability in
soils. Plants have developed subtle strategies to adapt to P deficiency,
such as changing their root system architecture, enhancing acquisition
of P from the environment and recycling internal P. These strategies in-
volve a number of metabolic genes, transcription factors, P transporters,
hormones and miRNAs [1,7–9]. Soybean plants have also adopted some
strategies to adapt to P deficiency; including changes of root morpholo-
gy and architecture, enhancement of root symbiosis and induction of
root exudates [10]. Variety numbers of molecular elements involved in
these pathways have been identified. More than 200 genes were identi-
fied to respond to P starvation in the roots and shoots of soybean seed-
lings. One of these genes, GmEXPB2 (Glycine max β-expansins), can
enhance both P utilization efficiency and P responsiveness by regulating
adaptive changes of the root system architecture [9,11]. A total of 44
phosphate-starvation responsive proteins were identified in soybean
nodules after depletion of phosphate, and interestingly, adaptive re-
sponses to P starvation occur differently between roots and shoots
[12]. Overexpression of an Arabidopsis purple acid phosphatase (PAP)
gene AtPAP15 increased the secretion of APase (acid phosphatase)
from transgenic soybean plants, significantly enhanced intracellular
APase activity in leaves and P utilization efficiency and yield under

http://dx.doi.org/10.1016/j.bbapap.2016.02.001
1570-9639/© 2016 Elsevier B.V. All rights reserved.
428 A. Sha et al. / Biochimica et Biophysica Acta 1864 (2016) 427–434
conditions of low organic P [13]. The expression of 23 GmPAPs was in-
duced or enhanced by P starvation in different soybean tissues [14].
Additionally, the expression of 110 miRNAs differed in roots or shoots
in soybeans under P deficient and adequate conditions [15].
In previous studies, the soybean cultivar BX10 has been identified as
a P-efficient genotype [16,17] and a number of early or late P-starvation
responsive genes and miRNAs were identified from BX10 based on the
transcriptional expression profiles and deep sequencing [9,15]. In this
study, a proteomic approach was utilized to investigate the protein ex-
pressional patterns in the shoots and roots of BX10 under P deficient
(low P) and sufficient conditions (high P). As no candidate early P star-
vation responsive genes were identified in the shoots under P deficiency
treatment for 0.5 h to 12 h [9] and the most tested miRNAs were differ-
entially expressed at 3 days (d) and 6 d under P deficiency [15], time
points of 3 d and6 d were selected for this study. The objective was to
identify P deficiency responsive proteins in soybean to further explain
the molecular mechanisms of P acquisition efficiency.
2. Materials and methods
2.1. Plant material
The seeds of P-efficient soybean cultivar BX10 were surface-
sterilized with 0.1% HgCl2 for 10 min. After five rinses with sterilized
distilled water, the seeds were germinated on wet paper tissue for one
week at 25 °C. Uniformly germinated seedlings were transplanted
into tanks (50 cm × 40 cm × 15 cm) with 1/2 modified Hoagland nutri-
ent solution and were grown in a growth chamber at temperatures of
26/20 °C (day/night), photo flux density of 480 μM m−2 s−1, a 16 h pho-
toperiod and relative humidity of 70%. The nutrient solution was re-
placed every 3 days. When the first trifoliate leaf was fully developed,
the seedlings were treated with low or high P solution including
0.2 μM KH2PO4 or 1000 μM KH2PO4, respectively, in the 1/2 modified
Hoagland nutrient solution. Treatments were carried out in triplicate
tanks with 30 seedlings each. The shoots (leaves together with stems)
and roots of five plants at each time point (3 d, 6 d) were collected
and served as one replicate, with three replicates for each treatment.
2.2. Protein extraction
Three biological replicates were conducted for the proteomic analy-
sis. For each biological replicate, shoots and roots were pooled from five
soybean seedlings. Proteins were extracted from the shoots or roots fol-
lowing the protocol developed by Damerval et al. [18] with the follow-
ing modifications. About 2.0 g shoots or roots were quickly ground with
a mortar in liquid nitrogen. The sample powder was suspended in 10%
w/v trichloroacetic acid/acetone with 0.07% v/v 2-mercaptoethanol
and incubated for 2 h at − 20 °C. After centrifugation, the pellet was
washed with cold acetone containing 0.07% v/v 2-mercaptoethanol to
remove pigments and lipids until the pellet was colorless. Proteins
were dried under a vacuum and resuspended in 1 mL rehydration buff-
er: 9.5 M urea, 5 mM K2CO3, 1.25% sodium dodecyl sulfate (SDS), 0.5%
dithiothreitol, 2% LKB Ampholines, pH 3.5 to 10, and 6% Triton X-100.
The resuspended proteins were sonicated with 4 cycles of 0.8 s open
and 0.8 s closed and repeated once after cooled on ice. The soluble pro-
teins from the supernatant were centrifuged for 20 min at 12,000 rpm at
4 °C and collected. The protein concentrations of the samples were
quantified via a Bradford assay [19].
2.3. 2-D electrophoresis
An Ettan IPGphor3 apparatus (GE Healthcare) was used for isoelec-
tric focusing (IEF) with immobilized pH-gradient (IPG) strips (pH 4.0–
7.0 linear gradient, 24 cm). After the IPG strips were rehydrated,
1200 μg of protein was loaded for IEF with the following program:
300 V for 0.5 h, 700 V for 0.5 h, 1500 V for 1.5 h, 9000 V for 3 h and
9000 V for 4 h. The total voltage was 54 kVh. After IEF electrophoresis,
the strips were equilibrated with the equilibration buffer, containing
1% w/v DTT, 6 M urea, 30% w/v glycerol, 2% SDS 50 mM Tris–HCl
(pH 8.8) and 0.002% bromophenol blue. After 15 min of equilibration,
the strips were incubated in the same equilibration buffer containing
2.5% iodoacetamide for another 15 min. The equilibrated strips were
used for the second dimension, which was 12.5% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in an Ettan
DALTsix electrophoresis unit (GE Healthcare). After SDS-PAGE, the
gels were stained with Coomassie brilliant blue R-250 and scanned
with an UMAX PowerLook 1100 scanner. Total protein spots in the
gels were subsequently analyzed and counted with the ImageMaster
2D platinum 5.0 software (GE Healthcare). All the spots exhibiting
differential accumulations of more than 2.0-fold (P b 0.05) were consid-
ered to be differentially expressed proteins. For each treatment, protein
extraction and 2-D electrophoresis were repeated three times.
2.4. In-gel digestion
Protein spots which changed the abundances during treatment were
excised from gels, washed with Millipore-pure water twice, destained
twice with 60 μL of 50 mM NH4HCO3 and 50% acetonitrile (ACN) and
then dried twice with 60 μL of ACN, followed by soaking in ice-cold
digestion solution (trypsin 12.5 ng/mL and 20 mM NH4HCO3) for
20 min. This mixture was then transferred into an incubator at 37 °C
for overnight digestion. Finally, peptides in the supernatant were col-
lected after two extractions with 60 μL extraction solution (5% formic
acid in 50% ACN).
2.5. MALDI-TOF-TOF MS analysis
The peptide solution described above was dried under nitrogen, and
resolubilized in 2 μL of matrix solution (α-cyano-N-hydroxycinnamic
acid). The mixture was then spotted on a MALDI target plate (Applied
Biosystems, USA). MALDI-TOF-TOF MS analyses were conducted with
a 4800 Plus MALDI-TOF-TOF™ analyzer (Applied Biosystems). The UV
laser was operated at a 200-Hz repetition rate with a wavelength of
355 nm. The accelerated voltage was operated at 20 kV and the mass
resolution was maximized at 1500 Da. Myoglobin digested with trypsin
was used to calibrate the instrument under internal calibration mode.
All acquired spectra of samples were processed with 4800 Plus TM
Software (Applied Biosystems) in the default mode. The data were
searched by GPS Explorer (V 3.6) with the search engine MASCOT
(V 2.1) against the NCBInr database and UniProt database (Version of
2015_03). The search was performed by selecting the Glycine max tax-
onomy. The other parameters were as follows: peptide molecular
mass ranged from 800 to 4000 Da, with one missing cleavage, MS toler-
ance of 50 ppm, MS/MS tolerance of 0.6 Da, fixed modifications of
carbamidomethyl (Cys) and variable modifications of oxidation (Met),
proteins with scores greater than 72 or a best ion score (MS/MS) of
more than 30 were considered significant. Only spots with statistical
significance (Student's t test, p b 0.05) and reproducible changes were
considered. The protein spots with an abundance change ratio of at
least two were selected as differentially expressed proteins.
2.6. Quantitative reverse transcription-polymerase chain reaction
(qRT-PCR)
qRT-PCR analysis was performed to quantify the transcriptional
levels of genes. Total RNA was extracted with Trizol (Invitrogen,
Carlsbad, CA, USA) from the samples of soybean seedling shoots and
roots and digested with DNase I (Ambion, USA) to eliminate genomic
DNA contamination. A total of 2 μg of RNA was used in cDNA synthesis
according to the manufacturer's instructions (Promega, USA). The quan-
tity and quality of isolated total RNA was examined by spectrophotom-
eter and gel electrophoresis. The qRT-PCR reaction was performed with
 A. Sha et al. / Biochimica et Biophysica Acta 1864 (2016) 427–434 429

the Roter-Gene Q Real-Time PCR System (Qiagen, Germany). The SYBR

GreenMasterMix was used to identify mRNA level according to
the manufacturer's instructions (Tiangen, China). To design RT-PCR
primers, the sequence of each differentially expressed protein was
first used as a tBLASTn search term against soybean ESTs (http://
compbio.dfci.harvard.edu). Then the EST was used as a BLASTN term
against the soybean genome (http://www.phytozome.net). The
primers were designed based on the corresponding genome sequences
(Table S1). All gene expression analyses were performed in biological
triplicate. Relative expression levels were calculated from the ratio of
expression levels of candidate genes to that of the housekeeping gene
actin. Amplification was performed in a volume of 10 μL containing
2 μL cDNA, 5 μL SuperReal PreMix (Tiangen) and 1 μM forward and re-
verse primers. The PCR program was as follows: an initial polymerase
activation step for 15 min at 94 °C, 40 cycles of denaturation for 15 s
at 94 °C, annealing for 10 s at 57 °C and extension for 25 s at 72 °C.
The melting course was ramped from 50 °C to 90 °C in 1 °C increments,
and waiting for 90 s of pre-melt conditioning on first step and 5 s for
each step afterwards.
Fig. 2. Identification of proteins responsive to P deficiency. All proteins with increased and
decreased levels under low P are indicated by the arrows with numbers. The spot number
3. Results
referred to the corresponding numbers in Table1. Spots C, D, E, F, I, J, K and L represented
the proteins with increasing accumulation in roots at 3 d under high P, roots at 3 d under
3.1. Identification of the P deficiency responsive proteins low P, roots at 6 d under high P, roots at 6 d under low P, shoots at 3 d under high P, shoots
at 3 d under low P, shoots at 6 d under high P, and shoots at 6 d under low P, respectively.
The results from the representative 2-DE gels showed a distinct and
reproducible separation of proteins, in which more than 700 protein proteins were identified from roots and shoots, respectively.). Spot F4
spots were observed (Fig. 1, Fig. S1–4). Compared with the 2-D protein was removed although it was identified by MALDI TOF/TOF MS but it
profile at high P conditions, a total of 98 protein spots exhibited greater is not significant (Table 1). Among the 85 protein spots, F42, J15, and
than 2.0-fold abundance changes in roots or shoots at low P treatments L5 are identified to correspond to multiple proteins and several spots
(Fig. 2). Following analysis by MALDI TOF/TOF MS, 85 protein spots were identified as identical proteins by searching against the NCBInr da-
were successfully identified to correspond to 88 proteins (51 and 37 tabase and UniProtKB database. The identical proteins include C24 and

Fig. 1. Representative 2-DE gels of proteins from shoots and roots under low phosphorus (P) conditions. a, low P treated shoots at 3 d; b, low P treated shoots at 6 d; c, low P treated roots at
3 d; and d, low P treated roots at 6 d. The seedlings of soybean cultivar BX10 were treated under low P (0.2 μM KH2PO4) for 3 d or 6 d and the shoots and roots were harvested for analysis of
2-DE. The numbers with arrows indicate the differentially expressed proteins and identified protein spots.
430 A. Sha et al. / Biochimica et Biophysica Acta 1864 (2016) 427–434

Table 1
Differentially accumulated proteins identified in shoots and roots of P-efficient soybean BX10 under P stresses.
a b c d e f g h i
Spot Accesion no. Protein name TMr /EMr TpI/EpI Coverage Score Specificity Time
number kDa/kDa (%) −P:+P

Shoot
Carbohydrate metabolism
I10 gi|4930130 Chain A, D-ribulose-5-phosphate 3-epimerase, 24.77/22.72 5.75/6.05 8 99 1:8.9 3d
Solanum tuberosum chloroplasts
K4 gi|2506277 RuBisCO large subunit-binding protein subunit beta, chloroplastic 63.28/55.58 5.85/5.20 11 184 1:2.4 6d
K7 gi|2274838 Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, 47.94/51.19 6.30/5.61 12 127 1:2.5 6d
Zelkova serrata
K5 gi|351724891 Enolase, Glycine max 47.97/51.29 5.31/5.50 29 338 1:2.8 6d
K8 gi|351724891 Enolase, Glycine max 47.97/51.82 5.31/5.49 32 448 Only +P 6d
L5-1 gi|351724891 Enolase, Glycine max 47.97/34.00 5.31/4.55 15 83 2.4:1 6d
K12 gi|5929964 Malate dehydrogenase, Glycine max 36.34/38.34 8.23/6.23 28 259 1:2.0 6d
J15–1 gi|5929964 Malate dehydrogenase, Glycine max 36.35/24.66 8.23/4.50 22 122 5.2:1 3d
L11 gi|351723339 Endochitinase PR4, Glycine max 26.25/24.34 4.90/4.49 16 250 2.0:1 6d
L12 gi|351725619 Cupin family protein, Glycine max 22.01/17.00 5.21/5.58 38 404 2.2:1 6d
Energy metabolism
J2 gi|13937039 Cytosolic glutamine synthetase alpha, Glycine max 9.06/37.25 8.31/5.27 45 252 2.3:1 3d
J14 gi|351724927 Chlorophyll a/b-binding protein, Glycine max 27.88/23.95 5.14/4.83 8 95 3.1:1 3d
J17 gi|255640161 Ribose-5-phosphate isomerase, Glycine max 29.89/24.53 5.37/4.71 16 141 2.0:1 3d
J18 gi|255640161 Ribose-5-phosphate isomerase, Glycine max 29.89/23.03 5.37/4.80 16 160 2.6:1 3d
K26 gi|255645102 Chaperonin, Glycine max 26.64/23.55 6.77/5.40 47 328 1:2.3 6d
K29 gi|255638460 ATP synthase epsilon chain, chloroplastic, Glycine max 8.10/17.00 5.02/5.20 48 173 1:2.7 6d
L2 gi|91214148 ATP synthase CF1 alpha subunit, Glycine max 55.77/51.93 5.15/5.15 30 466 2.1:1 6d
L3 gi|91214148 ATP synthase CF1 alpha subunit, Glycine max 55.77/52.68 5.15/5.09 28 490 2.9:1 6d
L8 gi|195657267 Cytochrome c oxidase subunit, Zea mays 18.46/25.35 4.35/4.50 20 192 2.5:1 6d
Cellular processes
L5-2 gi|255644930 Peroxidase, Glycine max 34.79/34.00 5.01/4.55 24 123 2.4:1 6d
J8 gi|182375363 Mucunain, Mucuna pruriens 47.84/27.15 6.41/4.74 6 98 2.1:1 3d
J12 gi|351724281 Cysteine protease-like, Glycine max 39.70/25.62 7.01/4.76 4 114 2.2:1 3d
L14 gi|351734390 Actin depolymerizing factor 1, Glycine max 16.08/17.00 6.15/5.93 28 193 3.1:1 6d
Defense/interaction with environment
J15–2 gi|351724171 Alpha-amylase/subtilisin inhibitor,Glycine max 23.20/24.66 4.47/4.50 18 136 5.2:1 3d
K17 gi|351725349 Alpha-amylase/subtilisin inhibitor, Glycine max 23.67/35.20 5.52/4.96 7 94 1:2.5 6d
L10 gi|351725349 Alpha-amylase/subtilisin inhibitor, Glycine max 23.67/24.79 5.52/4.55 7 86 3.0:1 6d
L13 gi|229597555 Chain A, Nmr solution structure of soybean allergen Gly M 4, 16.63/17.00 4.69/4.50 45 157 2.4:1 6d
Glycine max
L15 gi|351724283 Ripening related protein, Glycine max 17.72/17.00 5.38/5.21 51 130 3.0:1 6d
L17 gi|205829383 Profilin, pollen allergen beta v 2 2.52/17.00 4.03/4.50 56 93 2.6.1 6d
Nucleotide and amino acid metabolism
I7 gi|255642364 Fumarylacetoacetase, Glycine max 46.12/40.40 5.95/5.85 30 189 1:2.0 3d
Secondary metabolism
I8 gi|3334150 Magnesium-chelatase subunit chlI, Chloroplastic 46.07/38.07 5.49/4.91 38 275 1:2.5 3d
K19 gi|255637531 Isoflavone reductase like, Glycine max 34.29/34.00 5.73/5.89 21 320 1:2.2 6d
Signal transduction
I17 gi|351724251 Translationally-controlled tumor protein homolog, Glycine max 19.10/17.00 4.57/4.70 26 197 1:2.2 3d
Other metabolism process
J1 gi|255578278 Pro-resilin, Ricinus communis 41.39/47.06 4.65/4.66 6 85 3.2:1 3d
L7 gi|351722347 Nascent polypeptide-associated complex subunit alpha-like 22.11/26.08 4.43/4.50 28 123 2.3:1 6d
protein, Glycine max
K27 gi|351725517 Nascent polypeptide-associated complex subunit beta, Glycine max 17.51/19.09 7.90/6.51 21 100 1:66.6 6d
L16 gi|351723479 Nesprin-1, Glycine max 17.36/17.00 9.56/5.58 15 135 3.57:1 6d

Root
Protein biosynthesis/processing
F49 gi|351721220 Eukaryotic translation initiation factor 5A3,Glycine max 17.68/17.00 5.60/5.78 35 212 2.7:1 6d
D27 gi|255552604 Peptidyl-prolyl cis-trans isomerase, Ricinus communis 51.54/37.20 4.97/4.56 18 180 2.9:1 3d
D31 gi|255552604 Peptidyl-prolyl cis-trans isomerase, Ricinus communis 51.54/34.24 4.97/4.64 14 210 Only −P 3d
D9 gi|355513660 Ubiquilin, Medicago truncatula 57.67/55.47 4.73/4.76 16 156 2.7:1 3d
D11 gi|355513660 Ubiquilin, Medicago truncatula 57.67/54.21 4.73/4.71 14 97 2.8:1 3d
E18 gi|355513660 Ubiquilin, Medicago truncatula 57.67/34.85 4.73/4.98 14 97 Only +P 6d
D21 gi|255641336 26S proteasome non-ATPase regulatory subunit 4-like isoform 2, 43.06/47.54 4.62/4.47 29 175 3.1:1 3d
Glycine max
F14 gi|255570990 Heat shock protein, Ricinus communis 75.43/72.00 5.35/4.92 16 354 4.9:1 6d
F15 gi|255570990 Heat shock protein, Ricinus communis 75.43/72.00 5.35/4.94 13 327 3.9:1 6d
F16 gi|255570990 Heat shock protein, Ricinus communis 75.43/72.00 5.35/4.96 14 402 2.9:1 6d
F17 gi|255570990 Heat shock protein, Ricinus communis 75.43/72.00 5.35/4.98 18 363 2.0:1 6d
F19 gi|211906494 Heat shock protein 70, Gossypium hirsutum 71.27/66.24 5.14/4.98 17 275 2.0:1 6d
D8 gi|226500540 Heat shock 70 kDa protein, Vitis vinifera 72.99/59.61 5.62/5.42 6 79 3.4:1 3d
D16 gi|255644546 DNA repair protein RAD23–3, Glycine max 41.23/44.84 4.80/4.74 9 163 2.4:1 3d
D19 gi|255644546 DNA repair protein RAD23–3, Glycine max 41.23/45.45 4.80/4.90 14 268 3.9:1 3d
D25 gi|255644546 DNA repair protein RAD23–3, Glycine max 41.23/41.59 4.80/4.70 13 210 Only −P 3d
Energy metabolism
D13 gi|118429132 Vacuolar ATPase subunit B, Kalidium foliatum 54.17/46.58 4.93/4.93 31 312 Only −P 3d
D14 gi|118429132 Vacuolar ATPase subunit B, Kalidium foliatum 54.17/48.73 4.93/5.00 26 288 9.0:1 3d
D18 gi|118590578 F0F1 ATP synthase subunit beta, Stappia ggregate IAM 12614 50.81/48.73 4.77/5.00 9 126 2.2:1 3d
D23 gi|217072994 Actin, Medicago trunculata 41.76/40.58 5.31/5.39 33 251 3.8:1 3d
 A. Sha et al. / Biochimica et Biophysica Acta 1864 (2016) 427–434 431

Table 1 (continued)
a b c d e f g h i
Spot Accesion no. Protein name TMr /EMr TpI/EpI Coverage Score Specificity Time
number kDa/kDa (%) −P:+P

Root
Energy metabolism
D37 gi|255627487 Cytochrome b6-f complex iron–sulfur subunit, Glycine max 24.54/17.00 8.67/5.61 34 232 2.1:1 3d
E3 gi|159466892 Beta subunit of mitochondrial ATP synthase, Chlamydomonas reinhardtii 61.95/48.03 4.99/4.94 5 157 Only +P 6d
E10 gi|125525665 S-adenosylmethionine synthase 2, Oryza sativa 23.00/44.52 8.85/5.67 30 147 1:3.9 6d
E12 gi|13937039 Cytosolic glutamine synthetase alpha, Glycine max 9.06/37.20 8.31/5.15 45 143 1:3.4 6d
F36 gi|10946357 Cytosolic glutamine synthetase GSbeta1, Glycine max 39.14/31.28 5.48/5.11 29 85 3.1:1 6d
Cellular processes
C24 gi|351734390 Actin-depolymerizing factor 2, Glycine max 16.08/17.00 6.15/6.15 26 262 1:11.8 3d
D33 gi|355486571 Fructokinase, Medicago truncatula 35.38/34.00 5.20/4.95 17 124 2.1:1 3d
D35 gi|351722933 Adenine phosphoribosyl transferase, Trifolium repens 25.80/22.51 8.62/5.64 32 355 2.0:1 3d
E13 gi|255645037 Alpha-1,4-glucan protein synthase, Phaseolus vulgaris 42.05/38.23 5.66/5.67 26 162 1:4.0 6d
E19 gi|351724529 NADPH:isoflavone reductase, Glycine max 35.74/34.80 5.30/5.39 16 199 1:3.0 6d
E21 gi|351724529 NADPH:isoflavone reductase, Glycine max 35.74/34.80 5.30/5.46 28 199 Only +P 6d
E23 gi|255647567 Peroxidase 1, Sesbania rostrata 15.52/33.77 5.20/4.85 22 105 1:3.7 6d
F41 gi|182375363 Mucunain, Mucuna pruriens 47.85/27.28 6.41/4.76 6 88 2.1:1 6d
F42-1 gi|351725929 14–3-3 protein SGF14h, Glycine max 29.25/27.19 4.66/4.59 31 189 2.2:1 6d
Defense/interaction with environment
C25 gi|255647164 Nodulin-like protein, Glycine max 12.23/17.00 4.69/4.45 36 76 1:137.6 3d
E38 gi|351720849 Disease resistant response protein, Glycine max 20.52/19.71 5.10/5.00 31 222 1:4.0 6d
E40 gi|351720672 Trypsin inhibitor p20, Glycine max 22.86/17.00 5.39/5.25 24 186 1:5.7 6d
E41 gi|351723671 Trypsin inhibitor, Glycine max 18.25/17.02 6.12/4.98 22 113 1:2.9 6d
E42 gi|351723671 Trypsin inhibitor, Glycine max 18.25/17.00 6.12/5.09 44 172 1:2.3 6d
E43 gi|255630726 Kunitz trypsin inhibitor p20–1-like protein, Glycine max 22.61/17.00 4.83/4.44 11 76 1:241.1 6d
E45 gi|351726232 Stress-induced protein SAM22, Glycine max 16.70/17.00 4.80/4.50 60 158 1:9.8 6d
F50 gi|351726098 Ripening related protein, Glycine max 17.86/17.00 5.96/6.26 53 187 2.4:1 6d
Nucleotide and amino acid metabolism
D22 gi|255642364 Fumarylacetoacetase, Glycine max 46.12/44.25 5.95/5.88 23 160 2.2:1 3d
Secondary metabolism
D36 gi|351720734 Flavoprotein wrbA, Glycine max 21.79/21.70 6.09/6.06 26 236 2.2:1 3d
Other metabolism process
D4 gi|82469976 Patellin 1, Cucurbita pepo 67.03/94.15 4.68/4.15 2 72 3.5:1 3d
D5 gi|82469976 Patellin 1, Cucurbita pepo 67.03/94.15 4.68/4.87 1 47 2.5:1 3d
D26 gi|255646471 Ankyrin-repeat protein, Glycine max 38.02/41.47 4.53/4.50 14 212 2.5:1 3d
D32 gi|255627711 Nascent polypeptide-associated complex subunit 24.11/34.64 4.28/4.45 18 216 3.6:1 3d
alpha-like protein 2, Glycine max
E51 gi|108710322 Retrotransposon protein Ty1-copia subclass, Oryza sativa 15.23/17.00 5.50/5.20 16 100 1:4.6 6d
F42-2 gi|351723339 Endochitinase PR4, Glycine max 26.25/27.19 4.90/4.59 12 83 2.2:1 6d
C14 gi|224094919 Fructose-bisphosphate aldolase, Populus trichocarpa 38.63/29.75 5.89/6.48 15 189 1:7.3 3d
a
Numbering corresponds to the 2-DE gel in Fig. 2. Spots C, D, E, F, I, J, K and L represented the proteins with increasing accumulation in roots at 3 d under high P compared to low P, roots
at 3 d under low P compared to high P, roots at 6 d under high P compared to low P, roots at 6 d under low P compared to high P, shoots at 3 d under high P compare to low P, shoots at 3 d
under low P compared to high P, shoots at 6 d under high P compared to low P, and shoots at 6 d under low P compared to high P, respectively.
b
Accession number from the NCBI nr database.
c
Names and species of the proteins obtained via the MASCOT software from the NCBInr database.

L14, D4 and D5, D9, D11 and E18, D13 and D14, D16, D19 and D25, D27
and D31, D22 and I7, E19 and E21, E41 and E42, F14, F15, F16 and F17,
F41 and J8, E12, F36 and J2, J17 and J18, K5 and K8, K17 and L10, L2
and L3. Overall, a total of 61 distinct proteins were confidently identified
after removing the redundant proteins and the multiple protein spots
(Table 1). On the other hand, 37 unique proteins identified from root
after removing redundant proteins and multiple proteins including
spots D5, D9, D14, D19, D25, D31, E18, E19, E41, F15, F16, F17, F19,
and F36. 33 unique proteins identified from shoot after removing re-
dundant proteins and multiple proteins including spots J18, K8, L3,
and L10.
3.2. Differentially accumulated proteins in roots
A total of 51 proteins differentially accumulated in roots were cate-
gorized into six different groups based on their functional annotations.
The largest functional group was the protein biosynthesis/processing
group which accounts for 32%. The energy metabolism, cellular process-
es, and defense/interaction with environment groups followed with a
proportion of 18%, 16%, and 16%, respectively, and each one protein in-
volving in nucleotide metabolism and secondary metabolism (Table 1).
Furthermore, among 51 proteins, 33 of them were accumulated in root
after 3 days or 6 days P deficiency treatment, and 18 of 51 proteins were
accumulated in root after 3 days or 6 days P sufficient treatment.
3.3. Differentially accumulated proteins in shoots
The thirty-seven differentially accumulated proteins in shoots fell
into eight different groups includes carbohydrate metabolism, energy
metabolism, defense/interaction with environment processes, cellular
metabolism, nucleotide metabolism, secondary metabolism, signal
transduction, and other metabolism groups (Table 1). The largest func-
tional groups were the energy metabolism group accounting for 27%.
The carbohydrate metabolism, defense/interaction with the environ-
ment, and cellular processes groups followed with a proportion of
24%, 13%, and 10%, respectively. Two proteins are involved in secondary
metabolism, and one for each is involved in nucleotide metabolism,
signal transduction, respectively (Table 1). Furthermore, among 37 pro-
teins, 23 of them were accumulated in shoot after 3 days or 6 days P de-
ficiency treatment, and 14 of 37 proteins were accumulated in shoot
after 3 days or 6 days P sufficient treatment.
3.4. Consistent or inverse responses of protein family members to
P deficiency
Among the 61 identified proteins of unique function, 24 proteins
grouped to seven protein families with either consistent or inverse re-
sponse to P deficiency (Table 2). All members of the allergen family
432 A. Sha et al. / Biochimica et Biophysica Acta 1864 (2016) 427–434

Table 2
Consistent or inverse response of protein family members to P deficiency
a b c d e f
Spot number Accesion no. Proteins Specificity Time Tissue
−P: +P

L13 gi|229597555 Chain A, Nmr solution structure of soybean allergen Gly M 4 2.4:1 6d S
L17 gi|205829383 Profilin, pollen allergen beta v 2 2.6:1 6d S
F14,F15 gi|255570990 Heat shock protein 2.0 to 4.9: 1 6d R
F16,F17 6d R
F19 gi|211906494 Heat shock protein 70 2.0:1 6d R
D8 gi|226500540 Heat shock 70 kDa protein 3.4:1 3d R
L7 gi|351722347 Nascent polypeptide-associated complex subunit alpha-like protein 2.3:1 3d S
D32 gi|255627711 Nascent polypeptide-associated complex subunit alpha-like protein 2 3.6:1 3d R
K27 gi|351725517 Nascent polypeptide-associated complex subunit beta 1:66.6 6d S
J8 gi|182375363 Mucunain 2.1:1 3d S
J12 gi|351724281 Cysteine protease-like 2.2:1 3d S
F41 gi|182375363 Mucunain 2.1:1 6d R
L15 gi|351724283 Ripening related protein 3.0:1 6d S
F50 gi|351726098 Ripening related protein 2.4:1 6d R
D18 gi|118590578 F0F1 ATP synthase subunit beta 2.2:1 3d R
E3 gi|159466892 Beta subunit of mitochondrial ATP synthase Only +P 6d R
L2 gi|91214148 ATP synthase CF1 alpha subunit, 2.1:1 6d S
L3 gi|91214148 ATP synthase CF1 alpha subunit 2.9:1 6d S
E40 gi|351720672 Trypsin inhibitor p20 1:5.7 6d R
E41,E42 gi|351723671 Trypsin inhibitor 1:2.3 6d R
E43 gi|255630726 Kunitz trypsin inhibitor p20–1-like protein 1:241.1 6d R
a
Numbering corresponds to the 2-DE gel.
b
Accession number from the NCBI nr database or Plants EST database.
c
Names of the proteins obtained via the MASCOT software from the NCBInr database.
d
Specificity indicates the ratio of accumulation of a particular protein from roots or shoots under −P versus +P conditions.
e
Time indicates the ratios of accumulation of a particular protein from roots or shoots at 3 d or 6 d.
f
Tissue indicates a particular protein accumulated in roots (R) or shoots (S).

(spots L13, L17), heat shock protein family (spots F14, F15, F16, F17, F19,
D8), cysteine protease family (spots J8, J12, F41), trypsin inhibitor or
trypsin inhibitor-like proteins (spots E40, E41, E42, E43), and ripening
related protein family (L15, F50) showed consistent responses to P
deficiency, that is, the levels of members of these protein families
were elevated or decreased in roots or shoots at 3 d or 6 d under low
P conditions (Table 2). On the contrary, the members of ATP synthase
and nascent polypeptide-associated complex families exhibited inverse
responses to P deficiency, that is, the levels of members of these protein
families were either elevated or decreased in the roots or shoots at 3 d or
6 d under low P conditions (Table 2). The level of F0F1 ATP synthase
subunit beta (spot D18) was elevated in roots at 3 d, whereas the levels
of the β-subunit of mitochondrial ATP synthase (spot E3) and the two
ATP synthase CF1 alpha subunit (spots L2, L3) were decreased in roots
or increased in shoots at 6 d at low P level, respectively. The nascent
polypeptide-associated complex subunit alpha or alpha-like protein
(spots L7, D32) was increased in shoots or roots of 3 d but the nascent
polypeptide-associated complex subunit beta (spot K27) was decreased
in shoots of 6 d under low P conditions.
3.5. The temporal and spatial accumulation of P deficiency responsive
proteins
Among the 61 identified proteins of unique function, there were
only four proteins found in both shoots and roots in response to P defi-
ciency, at day 3 or 6.These four proteins included the cysteine protease
mucunain, actin depolymerizing factor 1, fumarylacetoacetase, and
cytosolic glutamine synthetase (Table 3). Mucunain was elevated in
both shoots at 3 d (spot J8) and roots at 6 d (spot F41) at low P level.
The actin depolymerizing factor 1 was up regulated in shoots (spot
L14), however down regulated in roots at 3 d (spot C24) at low P
level, whereas fumarylacetoacetase was down regulated in shoots
(spot I7) but up regulated in roots (spot D22) at 3 d at low P levels. Cy-
tosolic glutamine synthetase showed complex expressionex in roots at
3 d (spotsE12, F36) but decreased at 6 d (spot J2) at high P level.
3.6. qRT-PCR analyses of the encoded genes from significantly-changed
proteins
To explore whether the differentially expressed proteins were regu-
lated at transcriptional level and gain better insight to the temporal
expression of these genes, qRT-PCR was conducted to monitor
the time course expression of 18 genes from 3 d to 12 d (Table S1).
Twelve genes showed consistent accumulation patterns in both mRNA
and protein levels (Fig. 3, Table 1). Among them, Glyma14g09440,
Glyma09g30370, Glyma05g25810,Glyma17g07710 and Glyma12g36100
were up-regulated in shoots at 3 d or 6 d at low P level, corresponding
to the protein mucunain (spot J8), cytosolic glutamine synthetase
(spot J2), chlorophyll a/b-binding protein (J14), cytochrome c oxidase
subunit (L8) and ATP synthase CF1 alpha subunit (L2), respectively.
Glyma15g03430, Glyma12g00390, Glyma06g21290 and Glyma17g08020
were up-regulated in roots at 3 d or 6 d at low P level, corresponding
to fructokinase (spot D33), patellin 1 (spot D4), eukaryotic transla-
tion initiation factor 5A-4 (F49) and heat shock protein 70 (F19),
respectively. Glyma13g24050, Glyma08g18760, and Glyma12g19520
were down-regulated in shoots at 3 d or 6 d at low P level, which
corresponded to Magnesium-chelatase subunit chlI (I8), Rubisco large
subunit-binding protein subunit beta (K4) and malate dehydrogenase
(K12), respectively.
The transcriptional expression of six genes was not consistent
well with the accumulation of the corresponding proteins at the each
time point (Fig. 3, Table 1). The transcript expression of Glyma15g19580
did not change significantly in the shoots of 3 d between the low P
level and high P level, but the level of the corresponding protein
(cysteine protein-like, spot J12) was elevated more than 2-fold in
the shoots at 3 d at low P level. The transcriptional expression levels
of Glyma03g34830, Glyma11g12170 in shoots at 6 d under high P,
Glyma02g04740, Glyma19g01260 in roots at 3 d under low P, and
Glyma01g37810 in roots at 6 d under high P were decreased, but the ac-
cumulation of their corresponding proteins enclose (spot K5), Nascent
polypeptide-associated complex subunit beta (spot K27), ubiquilin
(spot D9), peptidyl-prolyl cis-trans isomerase (spot D27), and
 A. Sha et al. / Biochimica et Biophysica Acta 1864 (2016) 427–434 433

Table 3
The temporal and spatial accumulation of P deficiency responsive proteins.
a b c d e f
Spot number Accesion no. Proteins Specificity Time Tissue
−P: +P

J8 gi|182375363 Mucunain 2.1:1 3d S

F41 gi|182375363 Mucunain 2.1:1 6d R
L14 gi|351734390 Actin depolymerizing factor 1 3.1:1 3d S
C24 gi|351734390 Actin depolymerizing factor 1 1:11.8 3d R
I7 gi|255642364 Fumarylacetoacetase 1:2.0 3d S
D22 gi|255642364 Fumarylacetoacetase 2.2:1 3d R
E12 gi|13937039 Cytosolic glutamine synthetase alpha, Glycine max 1:3.4 6d R
F36 gi|13937039 Cytosolic glutamine synthetase alpha, Glycine max 3.1:1 6d R
J2 gi|13937039 Cytosolic glutamine synthetase alpha, Glycine max 2.3:1 3d S
a
Numbering corresponds to the 2-DE gel.
b
Accession number from the NCBI nr database.
c
Names of the proteins obtained via the MASCOT software from the NCBInr database.
d
Specificity indicates the ratio of accumulation of a particular protein from roots or shoots under −P versus +P conditions.
e
Time indicate the ratios of accumulation of a particular protein from roots or shoots at 3 d or 6 d.
f
Tissue indicates a particular protein accumulated in roots (R) or shoots (S).

NADPH:isoflavone reductase (spot E19) were elevated at the corre-
sponding time points, respectively.
4. Discussion
The present study attempted to identify proteins responsive to P de-
ficiency in the P efficient soybean cultivar BX10. A total of 88 proteins
and 61 unique proteins were identified to be responsive to P deficiency.
Among them, several proteins have been reported as P starvation
responsive proteins in previous study. These proteins include ribulose-
5-phosphate-3-epimerase in soybean (spot I10) [12]; enolase (spot
K5, K8, and L5-1), fructokinase (spot D33), ATP synthase (spot K29,
L2, L3, D18, and E3), RuBisco subunit binding protein (spot K4) in
maize [20]; cysteine synthase (spot J12) and peptidyl-prolyl cis-trans
isomerase in oilseed rape (spot D27, D31) [21]; malate dehydrogenase
(spot K12, J15-1) in soybean and maize [12,20]; glutamine synthetase
(spot J2, E12, and F36) in maize and rice [21,22]; and ATPase (spot
D13, D14 and D21), heat shock protein (spot D8, F14, F15, F16, F17,
and F19), and translation initiation factor 5A (spot F49) in maize and
oilseed rape [20,21], chitinase in rice (spot L11, F42-2) [22]. Some
genes corresponding to the identified proteins have also been demon-
strated to play a role in P signal pathway, such as the patellin gene fam-
ily (spot D4, D5). The accumulation of Arabidopsis PATL1 and PATL2
was changed slightly in P-depletion at protein level [23], but their ex-
pressions were altered greatly at transcriptional level [24]. However, a
significant amount of the proteins (40 proteins of 61 unique proteins
identified in this study) characterized in this study has never been pre-
viously reported in response to P starvation. The actual mechanisms of
these proteins involved in the P signal pathway needs to be investigated
further as the proteomic survey data are fragmentary and modification
of a complex subunit or of a protein does not mean modification of the
activity. Several genes corresponding to the identified proteins in this
study have been indicated to involve in abiotic regulation. A wheat
actin-depolymerizing factor regulates cold acclimation which alters

Fig. 3. qRT-PCR analyzing the transcript of genes encoding the identified Pi-starvation regulated proteins. The loaded mRNAs were normalized to the internal control actin gene and the
expression of the later time points was normalized to that at 3 d. LPR: low P treated roots; HPR: high P treated roots; LPS: low P treated shoots; and HPS: high P treated shoots. The error
bars represent SD from biological triplicates.
434 A. Sha et al. / Biochimica et Biophysica Acta 1864 (2016) 427–434
the cytoskeletal reorganization or cell wall components [25]. It is wor-
thy to investigate whether these proteins play cross-talking roles in
the abiotic response and P signaling pathway.
In this study, 37 and 33 unique proteins were identified from root
and shoot under high or low P condition, respectively. However, only
four proteins were both in root and shoot. It might indicate there are
different response and regulation mechanisms in different tissues
under high or low P condition in soybean. In addition, 70% differentially
expressed proteins belonged to metabolism in shoot, but only 35%
differentially expressed proteins belonged to metabolism in root.
More interesting, there were 31% differentially expressed proteins
belonged to protein biosynthesis or other protein processing in root
and no these kinds of proteins in shoot. It might suggest that once P sig-
nal started to transmit in root, more proteins were mobilized to involve
in this P signaling pathway.
A number of proteins showed either consistent or inverse response
to P deficiency (Table 2), and several proteins were accumulated in a
temporal and spatial way (Table 3). It is probable that these proteins
might function as early- or late-responsive proteins in shoots or roots
during P starvation, as two characterized responses to P starvation
exist in plant roots, that is, one is systemically controlled by whole-
plant P status and the other is governed by localized P signal [9].
qRT-PCR analyses of genes associated with identified proteins was in
agreement with protein dysregulations in most cases, validating the
protein changes upon P deficiency. In the few cases gene expression
data did not match the protein data. The discrepancy is likely caused
by the inconsistent expression for a specific gene at transcribe and
translation level.
5. Conclusions
In summary, our study identified a number of novel proteins
whose expression and abundance are significantly altered in response
to P starvation. For many of these proteins, changes in expression
were consistent with changes in expression of the corresponding
genes in soybean. This study provides a useful starting point for further
research that will provide a more comprehensive understanding of
molecular mechanisms through which soybean adapt to conditions of
P deficiency.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbapap.2016.02.001.
Conflict of interest statement
The authors have declared no conflict of interest.
Acknowledgements
This work was jointly supported by the Major S&T Projects on the
Cultivation of New Varieties of Genetically Modified Organisms (Grants
NO. 2011ZX08004-005), National Scientific and Technological Project
(Grants NO. 2011BAD35B06-4), and National Nonprofit Institute
Research Grant of CATAS-ITBB (Grant NO. 1610172011005). We are
grateful to Prof. Hai Nian, from the South China Agricultural University,
for kindly providing seeds of the BX10.
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