Identification of phosphorus deficiency responsive proteins in a high phosphorus acquisition soybean (Glycine max) cultivar through proteomic analysis

    Identification of phosphorus deficiency responsive proteins in a high phosphorus acquisition soybean (Glycine max) cultivar through proteomic analysis Aihua Sha, Ming Li, Pingfang Yang PII: DOI: Reference:

S1570-9639(16)30006-1 doi: 10.1016/j.bbapap.2016.02.001 BBAPAP 39695

To appear in:

BBA - Proteins and Proteomics

Received date: Revised date: Accepted date:

26 October 2015 7 January 2016 3 February 2016

Please cite this article as: Aihua Sha, Ming Li, Pingfang Yang, Identification of phosphorus deficiency responsive proteins in a high phosphorus acquisition soybean (Glycine max) cultivar through proteomic analysis, BBA - Proteins and Proteomics (2016), doi: 10.1016/j.bbapap.2016.02.001

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ACCEPTED MANUSCRIPT Identification of phosphorus deficiency responsive proteins in a high phosphorus acquisition soybean

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(Glycine max) cultivar through proteomic analysis

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Aihua Shaa * , Ming Lib, Pingfang Yangb*

a College of Agriculture, Yangtze University, Jingzhou, 434023, P.R China b Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical

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Garden, the Chinese Academy of Sciences, Wuhan 430074, China

*

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Corresponding author: Prof. Pingfang Yang, Key Laboratory of Plant Germplasm Enhancement

and Specialty Agriculture, Wuhan Botanical Garden, the Chinese Academy of Sciences, Wuhan

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430074, China

Fax: +86-27-87510956

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Tel: +86-27-87510956

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract As one of the major oil crops, soybean might be serious affected by phosphorus deficiency on

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both yield and quality. Understanding the molecular basis of phosphorus uptake and

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utilization in soybean may help to develop phosphorus (P) efficient cultivars. On this purpose, we conducted a comparative proteomic analysis on a high P acquisition soybean cultivar BX10 under low and high P conditions. A total of 61 unique proteins were identified as

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putative P deficiency responsive proteins. These proteins were involved in carbohydrate

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metabolism, protein biosynthesis/processing, energy metabolism, cellular processes, environmental defense/interaction, nucleotide metabolism, signal transduction, secondary

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metabolism and other metabolism related processes. Several proteins involved in energy

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metabolism, cellular processes, and protein biosynthesis and processing were found to be

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up-regulated in both shoots and roots, whereas, proteins involved in carbohydrate metabolism appeared to be down-regulated. These proteins are potential candidates for improving P

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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. Keywords Soybean; phosphorus efficiency; phosphorus deficiency; proteomic analysis

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ACCEPTED MANUSCRIPT Abbreviations: 2D, two-dimensional; ACN: acetonitrile; APase: Acid phosphatase; DTT, dithiothreitol; GmEXPB2: Glycine max β-expansins; GRF: General regulatory factor; IEF:

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isoelectric focusing; IPG: immobilized pH-gradient; EST: Expressed sequence tag;

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MALDI-TOF/TOF, matrix-assisted laser desorption/ionization time-of-flight-time-of-flight; MS, mass spectrometry; NADPH: Nicotinamide Adenine Dinucleotide Phosphate; P: Phosphorus; PAP: purple acid phosphatase; qRT-PCR: Quantitative real time - polymerase

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chain reaction; RAD23: Radiation sensitive23; RuBisCO: Ribulose bisphosphate carboxylase;

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SDS-PAGE: sodium dodecyl sulfate – polyacrylamide gel electrophoresis

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ACCEPTED MANUSCRIPT 1. Introduction Phosphorus (P) is an essential macronutrient for plant growth and development [1, 2]. As

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phosphorus is often deficient from soil or exists in unavailable forms that cannot be directly

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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 fertilizer improves crop production, but at the expense of causing severe environmental pollution and

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depletion of non-renewable P rocks [1, 5]. Therefore, it is necessary to develop

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environmentally – friendly and economically feasible strategies to improve crop production in soil with low P. One effective solution is to develop P-efficient cultivars based on the

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molecular understanding of P uptake and utilization in plants.

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Soybean (Glycine max (L.) Merr) is one of the most widely grown leguminous crops in the

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world, providing nutrition and oil for both human diet and animal husbandry [6]. However, the production and quality of soybean is seriously affected by the low P availability in soils.

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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 involve 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 morphology 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 identified to respond to P starvation in the roots and shoots of soybean seedlings. One of these genes, GmEXPB2 (Glycine max β-expansins), can enhance both P utilization efficiency and P 4

ACCEPTED MANUSCRIPT 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

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depletion of phosphate, and interestingly, adaptive responses to P starvation occur differently

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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

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efficiency and yield under conditions of low organic P [13]. The expression of 23 GmPAPs

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was induced 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

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adequate conditions [15].

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In previous studies, the soybean cultivar BX10 has been identified as a P – efficient genotype

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[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,

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15]. In this study, a proteomic approach was utilized to investigate the protein expressional patterns in the shoots and roots of BX10 under P deficient (low P) and sufficient conditions (high P). As no candidate early P starvation 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 differentially 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 5

ACCEPTED MANUSCRIPT 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

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paper tissue for one week at 25°C. Uniformly germinated seedlings were transplanted into

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tanks (50 cm × 40 cm × 15 cm) with 1/2 modified Hoagland nutrient solution and were grown in a growth chamber at temperatures of 26/20°C (day/night), photo flux density of 480 µM m-2s-1, a 16 h photoperiod and relative humidity of 70%. The nutrient solution was replaced

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every 3 days. When the first trifoliate leaf was fully developed, the seedlings were treated

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with low or high P solution including 0.2 μM KH2PO4 or 1,000 μM KH2PO4, respectively, in the 1/2 modified Hoagland nutrient solution. Treatments were carried out in triplicate tanks

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with 30 seedlings each. The shoots (leaves together with stems) and roots of five plants at

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each treatment.

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each time point (3 d, 6 d) were collected and served as one replicate, with three replicates for

2.2. Protein extraction

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Three biological replicates were conducted for the proteomic analysis. For each biological replicate, shoots and roots were pooled from five soybean seedlings. Proteins were extracted from the shoots or roots following the protocol developed by Damerval et al. [18] with the following 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 vacuum and resuspended in 1 mL rehydration buffer: 9.5 M urea, 5 mM K2CO3, 1.25 % sodium 6

ACCEPTED MANUSCRIPT dodecyl sulfate (SDS), 0.5 % dithiothreitol, 2 % LKB Ampholines, pH 3.5 to 10, 6 % Triton X-100. The resuspended proteins were sonicated with 4 cycles of 0.8 s open and 0.8 s

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closed and repeated once after cooled on ice. The soluble proteins from the supernatant were

samples were quantified via a Bradford assay [19]. 2.3. 2-D electrophoresis

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centrifuged for 20 min at 12000 rpm at 4 ℃ and collected. The protein concentrations of the

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An Ettan IPGphor3 apparatus (GE Healthcare) was used for isoelectric focusing (IEF) with

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immobilized pH-gradient (IPG) strips (pH 4.0-7.0 linear gradient, 24 cm). After the IPG strips were rehydrated, 1,200 µg of protein was loaded for IEF with the following program: 300 V

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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

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voltage was 54 KVh. After IEF electrophoresis, the strips were equilibrated with the

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equilibration buffer, containing 1% w/v DTT, 6M urea, 30% w/v glycerol, 2% SDS 50mM Tris-HCl (pH 8.8) and 0.002% bromophenol blue. After 15 min of equilibration, the strips

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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<0.05) were considered to be differentially expressed proteins. For each treatment, protein extraction and 2-D electrophoresis were repeated three times. 7

ACCEPTED MANUSCRIPT 2.4. In-gel digestion Protein spots which changed the abundances during treatment were excised from gels, washed

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with Millipore-pure water twice, destained twice with 60 µL of 50 mM NH4HCO3 and 50%

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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 20mM NH4HCO3) for 20 min. This mixture was then transferred into an incubator at 37°C for overnight digestion. Finally, peptides in the

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supernatant were collected after two extractions with 60 µL extraction solution (5% formic

2.5. MALDI-TOF-TOF MS analysis

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acid in 50% ACN).

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The peptide solution described above was dried under nitrogen, and resolubilized in 2 ul of

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matrix solution (α-cyano-N-hydroxycinnamic acid). The mixture was then spotted on a

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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

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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 1,500 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 taxonomy. The other parameters were as follows: peptide molecular mass ranged from 800 to 4,000 Da, with one missing cleavage, MS tolerance of 50 ppm, MS/MS tolerance of 0.6 Da, 8

ACCEPTED MANUSCRIPT 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

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considered significant. Only spots with statistical significance (Student’s t test, p < 0.05) and

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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)

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qRT-PCR analysis was performed to quantify the transcriptional levels of genes. Total RNA

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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

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DNA contamination. A total of 2 μg of RNA was used in cDNA synthesis according to the

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manufacturer's instructions (Promega, USA). The quantity and quality of isolated total RNA

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was examined by spectrophotometer and gel electrophoresis. The qRT-PCR reaction was performed with the Roter-Gene Q Real-Time PCR System (Qiagen, Germany). The SYBR

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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) 9

ACCEPTED MANUSCRIPT and 1 μM forward and reverse 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,

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annealing for 10 s at 57 °C and extension for 25 s at 72 °C. The melting course was ramped

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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.

3. Results

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3.1. Identification of the P deficiency responsive proteins

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The results from the representative 2-DE gels showed a distinct and reproducible separation of proteins, in which more than 700 protein spots were observed (Fig. 1, Fig. S1-4).

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Compared with the 2-D protein profile at high P conditions, a total of 98 protein spots

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exhibited greater than 2.0-fold abundance changes in roots or shoots at low P treatments (Fig.

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2). Following analysis by MALDI TOF/TOF MS, 85 protein spots were successfully identified to correspond to 88 proteins (51 and 37 proteins were identified from roots and

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shoots, respectively.). Spot F4 was removed although it was identified by MALDI TOF/TOF MS but it is not significant (Table 1). Among the 85 protein spots, F42, J15, and L5 are identified to correspond to multiple proteins and several spots were identified as identical proteins by searching against the NCBInr database and UniProtKB database. The identical proteins include C24 and 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 10

ACCEPTED MANUSCRIPT 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

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shoot after removing redundant proteins and multiple proteins including spots J18, K8, L3,

3.2. Differentially accumulated proteins in roots

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and L10.

A total of 51 proteins differentially accumulated in roots were categorized into six different

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groups based on their functional annotations. The largest functional group was the protein

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biosynthesis/processing group which accounts for 32%. The energy metabolism, cellular processes, and defense/interaction with environment groups followed with a proportion of

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18%, 16%, and 16%, respectively, and each one protein involving in nucleotide metabolism

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and secondary metabolism (Table 1). Furthermore, among 51 proteins, 33 of them were

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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.

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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 functional groups were the energy

metabolism

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accounting

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27%.

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metabolism,

defense/interaction with the environment, 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, 11

ACCEPTED MANUSCRIPT respectively (Table 1). Furthermore, among 37 proteins, 23 of them were accumulated in shoot after 3 days or 6 days P deficiency treatment, and 14 of 37 proteins were accumulated

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in shoot after 3 days or 6 days P sufficient treatment.

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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 response to P deficiency (Table 2). All members of

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the allergen family (spots L13, L17), heat shock protein family (spots F14, F15, F16, F17,

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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,

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F50) showed consistent responses to P deficiency, that is, the levels of members of these

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protein families were elevated or decreased in roots or shoots at 3 d or 6 d under low P

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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,

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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) were 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 12

ACCEPTED MANUSCRIPT Among the 61 identified proteins of unique function, there were only four proteins found in both shoots and roots in response to P deficiency, at day 3 or 6.These four proteins included

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the cysteine protease mucunain, actin depolymerizing factor 1, fumarylacetoacetase, and

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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

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level, whereas fumarylacetoacetase was down regulated in shoots (spot I7) but up regulated in

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roots (spot D22) at 3d at low P levels. Cytosolic glutamine synthetase showed complex expressionex in roots at 3 d (spotsE12, F36) but decreased at 6 d (spot J2) at high P level.

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3.6. qRT-PCR analyses of the encoded genes from significantly-changed proteins

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To explore whether the differentially expressed proteins were regulated at transcriptional level

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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

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showed consistent accumulation patterns in both mRNA and protein level (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 translation initiation factor 5A-4 (F49) and heat shock protein 70 (F19), respectively. Glyma13g24050, Glyma08g18760, and Glyma12g19520 were 13

ACCEPTED MANUSCRIPT 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

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(K4) and malate dehydrogenase (K12), respectively.

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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

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and high P level, but the level of the corresponding protein (cysteine protein-like, spot J12)

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was elevated more than 2-fold in the shoots at 3d at low P level. The transcriptional expression levels of Glyma03g34830, Glyma11g12170 in shoots at 6 d under high P,

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Glyma02g04740, Glyma19g01260 in roots at 3 d under low P, and Glyma01g37810 in roots at

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6 d under high P were decreased, but the accumulation of their corresponding proteins enclose

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(spot K5), Nascent polypeptide-associated complex subunit beta (spot K27), ubiquilin (spot D9), peptidyl-prolyl cis-trans isomerase (spot D27), and NADPH:isoflavone reductase (spot

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E19) were elevated at the corresponding time points, respectively.

4. Discussion

The present study attempted to identify proteins responsive to P deficiency 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 14

ACCEPTED MANUSCRIPT (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

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(spot J2, E12, and F36) in maize and rice [21, 22]; and ATPase (spot D13, D14 and D21), heat

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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 demonstrated to play a role in P

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signal pathway, such as the patellin gene family (spot D4, D5). The accumulation of

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Arabidopsis PATL1 and PATL2 was changed slightly in P-depletion at protein level [23], but their expressions were altered greatly at transcriptional level [24]. However, a significant

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amount of the proteins (40 proteins of 61 unique proteins identified in this study)

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characterized in this study have never been previously reported in response to P starvation.

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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

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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 the cytoskeletal reorganization or cell wall components [25]. It is worthy 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 mechanism in different tissues under high or low P condition in soybean. In addition, 70% differentially expressed proteins belonged to 15

ACCEPTED MANUSCRIPT 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

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biosynthesis or other protein processing in root and no these kinds of proteins in shoot. It

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might suggest that once P signal 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),

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and several proteins were accumulated in a temporal and spatial way (Table 3). It is probable

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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,

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one is systemically controlled by whole-plant P status and the other is governed by localized

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P signal [9].

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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

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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. 16

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Acknowledgements

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This work was jointly supported by the Major S&T Projects on the Cultivation of New

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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

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grateful to Prof. Hai Nian, from the South China Agricultural University, for kindly providing

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seeds of the BX10.

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2012, 55: 268-280.

SC R

[16] Dong D., Yan X., Peng X. Organic acid exudation induced by phosphorus deficiency and/or aluminium toxicity in two contrasting soybean genotypes. Physiol Plantarum 2004, 122: 190-199.

NU

[17] Xu Q.P., Luo C.Y., Liao H., Yan X.L., Nian H. Study on the response of soybean

MA

varieties to P deficiency. Soybean Sci 2003, 22:108-114.

[18] Damerval C., de Vienne D., Zivy M., Thiellement H. The technical improvements in

D

two-dimensional electrophoresis increase the level of genetic variation detected in

TE

wheat-seedling proteins. Electrophoresis 1986, 7:52-54.

CE P

[19] Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72: 248-254.

AC

[20] Li K., Xu C., Zhang K., Yang A., Zhang J. Proteomic analysis of roots growth and metabolic changes under phosphorus deficit in maize (Zea mays L.) plants. Proteomics 2007, 7:1501-1512.

[21] Yao Y., Sun H., Xu F., Zhang X., Liu S. Comparative proteome analysis of metabolic changes by low phosphorus stress in two Brassica napus genotypes. Planta 2011, 233:523-537. [22] Torabi S., Wissuwa M., Heidari M., Naghavi M., Gilany K., Hajirezaei M.R., Omidi M., Yazdi-Samadi B., Ismail A.M., Salekdeh G.H. A comparative proteome approach to decipher the mechanism of rice adaptation to phosphorous deficiency. Proteomics 2009, 9:159-170. 19

ACCEPTED MANUSCRIPT [23] Lan P., Li W., Schmidt W. Complementary proteome and transcriptome profiling in phosphate-deficient Arabidopsis roots reveals multiple levels of gene regulation. Mol Cell

IP

T

Proteomics 2012, 11: 1156-1166.

SC R

[24] Lin W.D., Liao Y.Y., Yang T.J., Pan C.Y., Buckhout T.J., Schmidt W. Coexpression-based clustering of Arabidopsis root genes predicts functional modules in early phosphate deficiency signaling. Plant Physiol 2011, 155: 1383-1402.

NU

[25] Ouellet F., Carpentier E., Cope M.J.T.V., Monroy A.F., Sarhan F. Regulation of a wheat

MA

actin-depolymerizing factor during cold acclimation. Plant Physiol 2001, 125: 360-368.

AC

CE P

TE

D

Tables

20

ACCEPTED MANUSCRIPT

IP

T

Table 1. Differentially accumulated proteins identified in shoots and roots of P-efficient soybean BX10 under P stresses. a

number

d b

Accesion no.

TMr /EMr

c

Protein name

CR

Spot

kDa/ kDa

Carbohydrate metabolism gi|4930130

K4

gi|2506277

K7

gi|2274838

K5

gi|351724891

Enolase, Glycine max

K8

gi|351724891

Enolase, Glycine max

L5-1

gi|351724891

Enolase, Glycine max

K12

gi|5929964

Malate dehydrogenase, Glycine max

J15-1

gi|5929964

Malate dehydrogenase, Glycine max

L11

gi|351723339

Endochitinase PR4, Glycine max

L12

gi|351725619

MA N

Chain A, D-Ribulose-5-Phosphate 3-Epimerase,

I10

TpI/EpI

Coverage (%)

g

Score

h

Specificity

i

Time

-P:+P

US

Shoot

f e

24.77/22.72

5.75/6.05

8

99

1:8.9

3d

63.28/55.58

5.85/5.20

11

184

1:2.4

6d

47.94/51.19

6.30/5.61

12

127

1:2.5

6d

47.97/51.29

5.31/5.50

29

338

1:2.8

6d

47.97/51.82

5.31/5.49

32

448

only +P

6d

47.97/34.00

5.31/4.55

15

83

2.4:1

6d

36.34/38.34

8.23/6.23

28

259

1:2.0

6d

36.35/24.66

8.23/4.50

22

122

5.2:1

3d

26.25/24.34

4.90/4.49

16

250

2.0:1

6d

Cupin family protein , Glycine max

22.01/17.00

5.21/5.58

38

404

2.2:1

6d

Solanum Tuberosum Chloroplasts

RuBisCO large subunit-binding protein subunit beta, chloroplastic

TE D

Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, Zelkova

AC

Energy metabolism

CE P

serrata

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 21

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

T

5.15/5.09

28

490

2.9:1

6d

L8

gi|195657267

Cytochrome c oxidase subunit, Zea mays

IP

ACCEPTED MANUSCRIPT

18.46/25.35

4.35/4.50

20

192

2.5:1

6d

34.79/34.00

5.01/4.55

24

123

2.4:1

6d

47.84/27.15

6.41/4.74

6

98

2.1:1

3d

39.70/25.62

7.01/4.76

4

114

2.2:1

3d

16.08/17.00

6.15/5.93

28

193

3.1:1

6d

23.20/24.66

4.47/4.50

18

136

5.2:1

3d

23.67/35.20

5.52/4.96

7

94

1:2.5

6d

23.67/24.79

5.52/4.55

7

86

3.0:1

6d

gi|255644930

Peroxidase , Glycine max

J8

gi|182375363

Mucunain, Mucuna pruriens

J12

gi|351724281

Cysteine protease-like, Glycine max

L14

gi|351734390

Actin depolymerizing factor 1, Glycine max

MA N

L5-2

US

CR

Cellular processes

Defense/interaction with environment gi|351724171

Alpha-amylase/subtilisin inhibitor，Glycine max

K17

gi|351725349

Alpha-amylase/subtilisin inhibitor, Glycine max

L10

gi|351725349

Alpha-amylase/subtilisin inhibitor , Glycine max

L13

gi|229597555

Chain A, Nmr solution structure of soybean allergen Gly M 4, Glycine max

16.63/17.00

4.69/4.50

45

157

2.4:1

6d

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

46.12/40.40

5.95/5.85

30

189

1:2.0

3d

gi|255642364

Fumarylacetoacetase, Glycine max

AC

I7

CE P

Nucleotide and amino acid metabolism

TE D

J15-2

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

Translationally-controlled tumor protein homolog, Glycine max

19.10/17.00

4.57/4.70

26

197

1:2.2

3d

Signal transduction I17

gi|351724251

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 protein, Glycine

22.11/26.08

4.43/4.50

28

123

2.3:1

6d 22

ACCEPTED MANUSCRIPT

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

17.68/17.00

5.60/5.78

35

212

2.7:1

6d

51.54/37.20

4.97/4.56

18

180

2.9:1

3d

51.54/34.24

4.97/4.64

14

210

only –P

3d

57.67/55.47

4.73/4.76

16

156

2.7:1

3d

57.67/54.21

4.73/4.71

14

97

2.8:1

3d

57.67/34.85

4.73/4.98

14

97

only +P

6d

CR

K27

IP

T

max

Root

US

Protein biosynthesis/processing gi|351721220

Eukaryotic translation initiation factor 5A3,Glycine max

D27

gi|255552604

Peptidyl-prolyl cis-trans isomerase, Ricinus communis

D31

gi|255552604

Peptidyl-prolyl cis-trans isomerase, Ricinus communis

D9

gi|355513660

Ubiquilin, Medicago truncatula

D11

gi|355513660

Ubiquilin, Medicago truncatula

E18

gi|355513660

Ubiquilin, Medicago truncatula

D21

gi|255641336

26S proteasome non-ATPase regulatory subunit 4-like isoform 2, Glycine max

43.06/47.54

4.62/4.47

29

175

3.1:1

3d

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

AC

CE P

TE D

MA N

F49

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 23

gi|217072994

Actin, Medicago trunculata

41.76/40.58

5.31/5.39

33

251

3.8:1

3d

D37

gi|255627487

Cytochrome b6-f complex iron-sulfur subunit, Glycine max

24.54/17.00

T

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

16.08/17.00

6.15/6.15

26

262

1:11.8

3d

35.38/34.00

5.20/4.95

17

124

2.1:1

3d

25.80/22.51

8.62/5.64

32

355

2.0:1

3d

42.05/38.23

5.66/5.67

26

162

1:4.0

6d

35.74/34.80

5.30/5.39

16

199

1:3.0

6d

35.74/34.80

5.30/5.46

28

199

only +P

6d

15.52/33.77

5.20/4.85

22

105

1:3.7

6d

47.85/27.28

6.41/4.76

6

88

2.1:1

6d

29.25/27.19

4.66/4.59

31

189

2.2:1

6d

12.23/17.00

4.69/4.45

36

76

1:137.6

3d

US

CR

D23

IP

ACCEPTED MANUSCRIPT

gi|351734390

Actin-depolymerizing factor 2, Glycine max

D33

gi|355486571

Fructokinase, Medicago truncatula

D35

gi|351722933

Adenine phosphoribosyl transferase, Trifolium repens

E13

gi|255645037

Alpha-1,4-glucan protein synthase, Phaseolus vulgaris

E19

gi|351724529

NADPH:isoflavone reductase, Glycine max

E21

gi|351724529

NADPH:isoflavone reductase, Glycine max

E23

gi|255647567

Peroxidase 1, Sesbania rostrata

F41

gi|182375363

Mucunain, Mucuna pruriens

F42-1

gi|351725929

14-3-3 protein SGF14h, Glycine max

CE P

Defense/interaction with environment

TE D

C24

MA N

Cellular Processes

gi|255647164

Nodulin-like protein, Glycine max

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

AC

C25

Nucleotide and amino acid metabolism 24

gi|255642364

Fumarylacetoacetase, Glycine max

46.12/44.25

5.95/5.88

23

160

2.2:1

3d

Flavoprotein wrbA, Glycine max

21.79/21.70

6.09/6.06

26

236

2.2:1

3d

67.03/94.15

4.68/4.15

2

72

3.5:1

3d

67.03/94.15

4.68/4.87

1

47

2.5:1

3d

38.02/41.47

4.53/4.50

14

212

2.5:1

3d

24.11/34.64

4.28/4.45

18

216

3.6:1

3d

15.23/17.00

5.50/5.20

16

100

1:4.6

6d

26.25/27.19

4.90/4.59

12

83

2.2:1

6d

38.63/29.75

5.89/6.48

15

189

1:7.3

3d

T

D22

IP

ACCEPTED MANUSCRIPT

Secondary metabolism D36

gi|351720734

D4

gi|82469976

Patellin 1, Cucurbita pepo

D5

gi|82469976

Patellin 1, Cucurbita pepo

D26

gi|255646471

Ankyrin-repeat protein, Glycine max

D32

gi|255627711

E51

gi|108710322

Retrotransposon protein Ty1-copia subclass, Oryza sativa

F42-2

gi|351723339

Endochitinase PR4, Glycine max

C14

gi|224094919

Fructose-bisphosphate aldolase, Populus trichocarpa

MA N

TE D

, Glycine max

Numbering corresponds to the 2-DE gel in Figure 2. Spots C, D, E, F, I, J, K and L represented the proteins with increasing accumulation in roots at 3 d under high

CE P

a

Nascent polypeptide-associated complex subunit alpha-like protein 2

US

CR

Other metabolism process

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

compared to high P, respectively.

AC

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

b

Accession number from the NCBI nr database .

c

Names and species of the proteins obtained via the MASCOT software from the NCBInr database.

25

ACCEPTED MANUSCRIPT Table 2. Consistent or inverse response of protein family members to P deficiency a

d

Spot

b

Accesion no.

e

-P : +P

gi|229597555

L17

gi|205829383

Profilin, Pollen allergen Beta v 2

gi|255570990

Heat shock protein

F19

gi|211906494

Heat shock protein 70

D8

gi|226500540

Heat shock 70 kDa protein

L7

gi|351722347

D32

gi|255627711

K27

gi|351725517

J8

gi|182375363

Mucunain

J12

gi|351724281

F41

Time

f

Tissue

2.4:1

6d

S

2.6:1

T

Chain A, Nmr Solution Structure Of

L13

6d

S

6d

R

6d

R

2.0:1

6d

R

3.4:1

3d

R

2.3:1

3d

S

3.6:1

3d

R

1:66.6

6d

S

2.1:1

3d

S

Cysteine protease-like

2.2:1

3d

S

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

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

1:241.1

6d

R

IP

Nascent polypeptide-associated complex subunit alpha-like protein

NU

Nascent polypeptide-associated

AC

E43

gi|255630726

SC R

2.0 to 4.9 :1

complex subunit alpha-like protein 2 Nascent polypeptide-associated

MA

complex subunit beta

Beta subunit of mitochondrial ATP synthase

CE P

F16,F17

Soybean Allergen Gly M 4

TE

F14,F15

a

Proteins

D

number

Specificity

c

Kunitz trypsin inhibitor p20-1-like protein

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. eTime indicate the ratios of accumulation of a particular protein from roots or shoots at 3 d or 6 d. f Tissue indicate a particular protein accumulated in roots (R) or shoots (S).

26

ACCEPTED MANUSCRIPT Table 3 The temporal and spatial accumulation of P deficiency responsive proteins

a

b

Accesion no.

c

d

Proteins

Specificity －P : +P

gi|182375363

Mucunain

2.1:1

F41

gi|182375363

Mucunain

2.1:1

L14

gi|351734390

Actin depolymerizing factor 1

C24

gi|351734390

Actin depolymerizing factor 1

I7

gi|255642364

Fumarylacetoacetase

D22

gi|255642364

Fumarylacetoacetase

F36

gi|13937039

J2

gi|13937039

alpha, Glycine max

NU

gi|13937039

Cytosolic glutamine synthetase alpha, Glycine max

MA

E12

Cytosolic glutamine synthetase

Cytosolic glutamine synthetase alpha, Glycine max

Time

f

Tissue

3d

S

6d

R

3.1:1

3d

S

1:11.8

3d

R

1:2.0

3d

S

2.2:1

3d

R

1:3.4

6d

R

3.1:1

6d

R

2.3:1

3d

S

SC R

J8

e

T

number

IP

Spot

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

AC

+ P conditions.

CE P

TE

D

a

e

Time indicate the ratios of accumulation of a particular protein from roots or shoots at 3 d or 6 d.

f

Tissue indicate a particular protein accumulated in roots (R) or shoots (S)

27

ACCEPTED MANUSCRIPT Figure Captions Fig. 1. Representative 2-DE gels of proteins from shoots and roots under low phosphorus (P)

T

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; d,

IP

low P treated roots at 6 d. The seedlings of soybean cultivar BX10 were treated under low P (0.2 µM

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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.

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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 referred to the

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corresponding numbers in Table1. Spots C, D, E, F, I, J, K and L represented the proteins with

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increasing accumulation in roots at 3 d under high P, roots at 3 d under low P, roots at 6 d under high P,

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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.

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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

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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; HPS: high P treated shoots. The error bars represent SD from biological triplicates.

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Figure 1

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Figure 2

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Figure 3

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ACCEPTED MANUSCRIPT Conflict of interest statement

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The authors have declared no conflict of interest.

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ACCEPTED MANUSCRIPT Highlights Proteomes of soybean under low and high phosphorus conditions were compared.

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1.

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2. 88 protein spots were identified as putative phosphorus deficiency responsive proteins, among

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them, 33 protein spots corresponding to 21 unique proteins have been reported as P starvation responsive proteins in previous study , and 55 protein spots corresponding to 40 proteins might be new

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identified proteins response to P starvation.

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