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Phosphoproteomic Dynamics of Chickpea (Cicer arietinum L.) Reveals Shared and Distinct Components of Dehydration Response Pratigya Subba, Pragya Barua, Rajiv Kumar, Asis Datta, Kamlesh Kumar Soni, Subhra Chakraborty, and Niranjan Chakraborty J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 01 Oct 2013 Downloaded from http://pubs.acs.org on October 2, 2013

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Journal of Proteome Research

Phosphoproteomic Dynamics of Chickpea (Cicer arietinum L.) Reveals Shared and Distinct Components of Dehydration Response

Pratigya Subba, Pragya Barua, Rajiv Kumar, Asis Datta, Kamlesh Kumar Soni, Subhra Chakraborty§ and Niranjan Chakraborty¶

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi110067, India.

RUNNING TITLE: Phosphoproteomic Dynamics of Dehydration Response

CORRESPONDING AUTHOR ¶

Dr. Niranjan Chakraborty National Institute of Plant Genome Research Aruna Asaf Ali Marg, New Delhi-110067, India. E-mail: [email protected] Tel: Fax:

00-91-11-26735178 00-91-11-26741658

§

Dr. Subhra Chakraborty National Institute of Plant Genome Research Aruna Asaf Ali Marg, New Delhi-110067, India. E-mail: [email protected] Tel: Fax:

00-91-11-26735186 00-91-11-26741658

1 ACS Paragon Plus Environment


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ABSTRACT Reversible protein phosphorylation is a ubiquitous regulatory mechanism that plays critical roles in transducing stress signals to bring about coordinated intracellular responses. To gain better understanding of dehydration response in plants, we have developed a differential phosphoproteome in a food legume, chickpea (Cicer arietinum L.). Three-week-old chickpea seedlings were subjected to progressive dehydration by withdrawing water and the changes in the phosphorylation status of a large repertoire of proteins were monitored. The proteins were resolved by 2-DE and stained with phosphospecific fluorescent Pro-Q Diamond dye. Mass spectrometric analysis led to the identification of 91 putative phosphoproteins, presumably involved in a variety of functions including cell defense and rescue, photosynthesis and photorespiration, molecular chaperones, and ion transport, among others. Multiple sites of phosphorylation were predicted on several key elements, which include both the regulatory as well as the functional proteins. A critical survey of the phosphorylome revealed a DREPP (Developmentally Regulated Plasma membrane Protein) plasma membrane polypeptide family protein, henceforth designated CaDREPP1. The transcripts of CaDREPP1 were found to be differentially regulated under dehydration stress further corroborating the proteomic results. This work provides new insights into the possible phosphorylation events triggered by the conditions of progressive water-deficit in plants.


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KEYWORDS: dehydration response; food legume; IMAC; mass spectrometry; posttranslational modification; phosphorylome dynamics

INTRODUCTION Dehydration is a serious threat to sustainable crop production worldwide. This is exemplified several times in regions where crop productivity graph is still largely dependent on rainfall statistics and ever-changing environmental conditions. Dehydration causes a great amount of loss, both in terms of biomass as well as economic returns. Therefore, studies on plant response to dehydration is crucial, specifically in the context of farming practices in developing world where a considerable proportion of potential crop biomass is lost due to water-deficit conditions. In recent years, extensive investigations at the molecular level on plant responses to environmental stress demonstrated the complexity of mechanisms involved in stress adaptation.1 However, the modus operandi of cellular machinery for the perception of stimuli and signal transduction has not been clearly understood. The observations that the expression of some genes are regulated by dehydration as well as alternative factors, whereas other genes respond only to single stimulus, indicate the existence of several signal transduction pathways in plants.2 Plants have developed various elaborate mechanisms to combat dehydration effect by rapidly modulating their proteome compositions through changes in their abundances and/or expression that are regulated by a number of potentially overlapping signal transduction networks that rely on post-translational modifications (PTMs) of many key cellular components.3 Therefore, characterization of the phosphorylome dynamics in plants would promote the understanding of core regulatory systems which eventually may help to improve agronomically important crop species. With the



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completion of genome sequencing of many crop species, the current major challenge is to determine the function of the genes, and that demands the identification of the encoded proteins and their modification. Phosphorylation is the most important PTM of proteins that modulate protein activity and transmit signals within cellular pathways and networks.4,5 The rapid finetuned control achieved through this modification is believed to be of considerable importance for regulating plants response to various environmental stresses.6 Protein phosphorylation is known to co-ordinate the synchronization of signal transduction through highly dynamic and complex networks. However, the transient stoichiometry of this modification and low abundance of most signaling proteins make the analysis complex.7 In recent years, the development of sensitive mass spectrometric techniques

in

conjunction

with

strategies

to

enrich

the

phosphorylated

peptides/proteins led to the identification of a large repertoire of phosphoproteins from several plant species.8-12 Most efforts in plant phosphoproteomic analyses have focused on the identification of protein phosphorylation in developmental stages13, cellular compartments14-16, chromatin structure17, and only recently has the focus extended to stress responses18-20. It is apparent that analysis of the temporal changes in protein phosphorylation will contribute in unraveling the role of specific phosphorylation events in key biological processes. Chickpea is one of the Neolithic founder crop assemblages and an important legume consumed as a source of vegetable protein throughout the world.21 It ranks third in terms of total global production22 and is the second most important rain-fed legume, cultivated mainly by small farmers in the semi-arid tropics, and West Asia and North Africa (WANA) region. Chickpea has garnered attention due to its resilience to adverse environmental conditions making it an interesting system to


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understand stress tolerance mechanisms.23-25 Its growth is limited mainly by the low moisture content in the soil, and dehydration tolerance is thus the most important ecophysiological traits for quantifying its actual harvest index. In this study, we catalogued the differentially regulated protein phosphorylation events in response to dehydration in chickpea seedlings. Several putative phosphoproteins involved in various biological functions were identified and inspected in silico for their sites of phosphorylation.

Furthermore,

IMAC

(Immobilized

Metal-ion

Affinity

Chromatography) enriched phosphorylated protein fraction led to the identification of several metabolism-related putative phosphoproteins. The differential expression profiles of the putative phosphoproteins may provide new insight into the underlying mechanisms and the involvement of these components in stress adaptation. This may also facilitate the targeted manipulation of metabolite routes for effective engineering strategies in chickpea for agricultural benefits.

EXPERIMENTAL PROCEDURES Plant Growth Condition and Stress Treatments Seeds of a relatively dehydration tolerant chickpea cultivar (Cicer arietinum L.cv. JG-62) were grown in a mixture of soil and soilrite (2:1, w/w; 10 plants/1.5liter-capacity pots) under controlled environmental conditions (25±2 °C, 50±5% relative humidity, 16 h photoperiod and 270 µmol m-2 s-1 light intensity)26. The pots were supplied with 100 mL water daily to maintain the soil moisture content to approximately 30%. A gradual dehydration condition was imposed on the 3-week-old seedling by withdrawing water i.e supply of water was stopped from 22nd day onwards. Unstressed and stressed seedlings were kept in parallel in the same growth room. The aerial part of the seedlings were harvested from unstressed and stressed



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seedlings at different time intervals up to 120 h and kept at -80 °C after quick-freezing in liquid nitrogen. The tissues from the unstressed seedlings were collected at each of the time-points during dehydration, and finally pooled to normalize the growth and developmental effects, if any. In a separate experiment, three-week-old seedlings were subjected to different treatments viz., NaCl, abscisic acid and methyl viologen by spraying the respective solutions. To mimic biotic stress response, the seedlings were treated with salicylic and jasmonic acid. Low temperature treatment was inflicted on the seedlings by keeping them at 4 °C. Tissues were instantly frozen in liquid nitrogen and stored at 80 °C. The entire process of plant growth and stress treatment was repeated thrice to generate three biological replicates.

Extraction of Proteins Tissues were ground to powder in liquid nitrogen and 2 g of the powder was transferred into pre-chilled 50 mL centrifuge tube containing 10 mL homogenization buffer [sucrose (40%), 50 mM HEPES-KOH (pH 7.5), beta-mercaptoethanol (1%), 1 mM EDTA (pH 7.5), 60 mM sodium fluoride, phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich)]. The homogenate was vortexed and mixed with 15 mL of Trisequilibrated phenol. The tubes were placed on a rocker platform at 4 °C for 30 min and then centrifuged (5000g, 15 min) for phase separation. The phenol phase was collected and precipitated overnight at -20 °C using 0.1 mM ammonium acetate in methanol. Protein pellets were recovered through centrifugation (10000g, 15 min) at 4 °C, and the resulting pellets were washed with 80% acetone, air dried and resuspended in rehydration buffer [8 M urea, 2 M thiourea and CHAPS (2%)].


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In-gel Kinase Assay In-gel kinase assay was carried out as described earlier.27 Briefly, 30 µg of protein were resolved onto 12.5% SDS-PAGE embedded with Myelin Basic Protein (MBP). After electrophoresis, SDS was removed with a washing buffer [25 mM Tris (pH 7.5), 0.5 mM DTT, 0.1 mM Na3VO4, 5 mM NaF, bovine serum albumin (0.05%) and Triton X-100 (0.1%)] three times for 30 min each. Renaturation of kinase/s was performed in the renaturation buffer [25 mM Tris (pH 7.5), 1 mM DTT, 0.1 mM Na3VO4 and 5 mM NaF] at 4 ºC overnight with three changes. The gel was then incubated in 30 mL of reaction buffer [25 mM Tris (pH 7.5), 2 mM EGTA, 10 mM MgCl2, 1 mM DTT, 0.1 mM Na3VO4, 200 µM ATP and γ32ATP] for 60 min. The reaction was terminated by transferring the gel into 5% TCA (w/v) containing 1% sodium pyrophosphate (w/v). Kinase activity was quantified by PhosphoImaging (Typhoon 9210, Molecular Dynamics). Molecular mass of the kinases was determined using pre-stained protein size markers (Bio-Rad Laboratories).

2-DE and Detection of Putative Phosphoproteins IEF was carried out with 1 mg protein per IPG strip (24 cm, pH 4-7) using IPGphor system (GE Healthcare). Proteins were diluted with 2-D rehydration buffer [8 M urea, 2 M thiourea, 2% CHAPS (w/v), 20 mM DTT, 0.5 % pharmalyte (v/v, pH 4-7) and 0.05% bromophenol blue (w/v)], and 450 µL of the diluted proteins was used to rehydrate the strips passively. Proteins were electrofocused up to 70000 VhT at 20 °C and the focused strips were subjected to reduction with DTT (1%, w/v) in 15 mL equilibration buffer [6 M urea, 50 mM Tris- HCl (pH 8.8), glycerol (30%, v/v) and SDS (2%, w/v)] followed by alkylation with iodoacetamide (2.5%, w/v) in the same buffer. The strips were then loaded onto 12.5% polyacrylamide gels and the proteins



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were resolved on Ettan Dalt-6 electrophoresis unit (GE Healthcare) at a constant voltage of 100 V. The electrophoresed proteins were stained to visualize the putative phosphoproteins and total proteins sequentially. The gels were treated with fixation solution overnight (50% methanol, 10% acetic acid) and then washed with deionized water (4 X 15 min) on an orbital shaker. The gels were stained with the modified ProQ Diamond staining8, followed by silver stain.

Image Acquisition and In-silico Analysis The Pro-Q Diamond stained gel images were captured with 532 nm excitation and 560 nm emission filters (Typhoon 9210, Molecular Dynamics) and the silverstained gels were scanned using a FluorS imaging system (Bio-Rad Laboratories). Three replicate gel images were computationally combined to generate ‘master gel’ for each time-point. A first level matchset was created using the ‘master gels’ of each of the time-points. Each spot included on the standard gel met with several criteria: the spot was present in at least two of the three gels, and qualitatively consistent in size and shape in the replicate gels. The “low quality” spots (with a quality score less than 30) were eliminated from further analysis. The remaining spot quantities were used to calculate the mean value for a given spot, which was then used as the spot quantity. The replicate gels used for making the first level matchset had at least a correlation coefficient value of 0.8. The spot densities were normalized against the total density in the gel image. The filtered spot quantities from the standard gels were assembled into a data-matrix of high quality spots to generate a second level matchset from each time-point. A Microsoft Excel compatible report was extracted and the


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normalized spot volume data was used for further analysis. A second normalization was done with a set of three unaltered spots identified across the time-points.

Peptide Preparation for Tandem Mass-Spectrometry

The candidate protein spots were mechanically excised from silver stained gels, in-gel digested with trypsin and peptides were extracted according to standard techniques28. Briefly, the excised gel plugs were washed twice with deionized water and destained using 100 mM sodium thiosulfate and 30 mM potassium ferricyanide solution (1:1, v/v) for 15 min. Three wash steps were performed in 50% acetonitrile (v/v) containing 25 mM NH4HCO3. The gel pieces were then dehydrated by soaking in 100% acid free acetonitrile (ACN) for 5 min and air-dried. Tryptic digestion was performed by incubating the gel pieces in 10 µL trypsin (10 ng/µL) overnight at 30 °C. Mass spectrometry was performed using the following instruments. For MALDI TOF/TOF analysis, the peptides were mixed with the matrix [αcyano-4-hydroxycinnamic acid, 1:1 (v/v)]. Data were acquired using 4800 MALDI TOF/TOF analyzer (AB Sciex). Protein identifications were based upon combined MS-MS/MS. MS spectra were obtained from 850 to 4000 m/z, for a total of 1500 laser shots operating at 355 nm and 200 Hz. MS/MS analyses were performed on suitable precursors using collision-induced dissociation (CID) with 1 kV collision energy and air as CID gas. Spectra were searched with the aid of GPS Explorer version 3.6 software

(AB

Sciex)

(www.matrixscience.com)

and against

a

licensed appropriate

version databases

of

Mascot

[NCBInr

2.1.0

(8994603

sequences; 3078807967 residues) or MSDB (3239079 sequences; 1079594700 residues)]. The following parameters were applied: taxonomy, viridiplantae; enzyme,



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trypsin; missed cleavage, 1; fixed modification, carbamidomethylation; variable modification, methionine oxidation; precursor mass tolerance, ±100 ppm; and peptide mass fragment tolerance, ±0.4 Da. In the final protein IDs (combined from PMF and MS/MS spectral data) the probability scores greater than the score fixed by Mascot were considered as positive identifications29,30 with p value

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