Comparative Phosphoproteome Analysis of the Developing Grains in

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Comparative Phosphoproteome Analysis of the Developing Grains in Bread Wheat (Triticum aestivum L.) under Well-Watered and WaterDeficit Conditions Ming Zhang,† Cao-Ying Ma,† Dong-Wen Lv,† Shou-Min Zhen, Xiao-Hui Li, and Yue-Ming Yan* College of Life Science, Capital Normal University, 100048 Beijing, China S Supporting Information *

ABSTRACT: Wheat (Triticum aestivum), one of the most important cereal crops, is often threatened by drought. In this study, water deficit significantly reduced the height of plants and yield of grains. To explore further the effect of drought stress on the development and yield of grains, we first performed a large scale phosphoproteome analysis of developing grains in wheat. A total of 590 unique phosphopeptides, representing 471 phosphoproteins, were identified under well-watered conditions. Motif-X analysis showed that four motifs were enriched, including [sP], [Rxxs], [sDxE], and [sxD]. Through comparative phosphoproteome analysis between well-watered and water-deficit conditions, we found that 63 unique phosphopeptides, corresponding to 61 phosphoproteins, showed significant changes in phosphorylation level (≥2-fold intensities). Functional analysis suggested that some of these proteins may be involved in signal transduction, embryo and endosperm development of grains, and drought response and defense under water-deficit conditions. Moreover, we also found that some chaperones may play important roles in protein refolding or degradation when the plant is subjected to water stress. These results provide a detailed insight into the stress response and defense mechanisms of developmental grains at the phosphoproteome level. They also suggested some potential candidates for further study of transgenosis and drought stress as well as incorporation into molecular breeding for drought resistance. KEYWORDS: Triticum aestivum, quantitative phosphoproteomics, grain development, drought stress, bioinformatics



INTRODUCTION

In recent years, studies of transcriptomics, proteomics, and metabolomics in wheat have revealed many clues to the mechanisms of seed development9−12 from different molecular perspectives. The development of seeds commonly occurs through a series of signal processes. Abscisic acid (ABA) is a key hormonal regulator that plays roles in seed maturation and stress tolerance.13 SnRK 2s, key kinases of the ABA signal system, are associated with the ABA signaling cascade and are required for the regulation of seed development and dormancy.14 In addition, receptor-like kinases (RLKs) found in many plants are associated with seed development and stress responses and are considered to be key regulators of plant growth and development.15,16 Terrestrial plants have developed specific responses and tolerance mechanisms to cope with adverse environments, particularly drought.17 Generally speaking, three distinct strategies to resist drought stress are available to plants: escape, avoidance, and tolerance.18 The drought response and tolerance mechanisms of plants tend to involve osmotic homeostasis or regulation, stress repair and defense (detox-

Wheat is a major cereal crop that is widely cultivated throughout the world because of its value as a major food source and its unique suitability to bread production. Its yield and quality remain a research priority for agricultural research. Wheat is often subjected to drought stress during its growth and development. Drought is one of the most studied abiotic stresses and significantly affects the yield of the world’s most important cereal staple crops, particularly wheat.1 Worldwide, most agricultural areas depend on rainfall alone (rather than irrigation), including almost all arid and semiarid areas, particularly in the Southern Hemisphere.2 In the North China Plain, most crops, particularly wheat, depend on rainfall and are exposed to drought stress during their development and maturation.3 Drought reduces the number of grains that develop and causes pollen sterility; these are the main causes of yield losses in wheat.4,5 In addition, the development of the embryo and endosperm during grain filling and maturing is generally sensitive to water stress, which affects the final yield of grains, both quantitatively and qualitatively.6−8 Therefore, it is critical to clarify the molecular mechanism of drought response and defense during wheat grain development. © 2014 American Chemical Society

Received: April 20, 2014 Published: August 22, 2014 4281

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in three biological replicates, were collected and stored at −70 °C prior to analysis.

ification), or growth inhibition.19 Previous studies on the transcriptomics and proteomics of grain development have identified some important genes and proteins related to drought acclimation and tolerance, which provided new insight into plant responses to drought stress and defense mechanisms against drought.20−23 To date, a number of techniques have been developed for the specific enrichment of phosphopeptides or phosphoproteins, such as immobilized metal affinity chromatography (IMAC) and TiO2-metal oxide affinity chromatography (MOAC),24 combined with label-free quantitative proteomic techniques.25,26 These technologies have accelerated phosphoproteomics research. Protein phosphorylation, one of the mostly studied posttranslational modifications, occurs through the ABA signal transduction pathway and acts on cellular growth and development and the stress response.27,28 Global phosphoproteomic studies on seed development in Arabidopsis thaliana, Vitis vinifera, Zea mays, and Glycine max have identified large numbers of phosphoproteins that are involved in signal transduction and grain development.29−32 Recently, a large-scale comprehensive study of phosphoproteome characteristics examined embryonic development of rice over time.33 To our knowledge, no phosphoproteome report has been released on developing grains in wheat, particularly under water stress. The recent release of the whole-genome shotgun sequencing for bread wheat and its progenitor A and D genomes34−36 are expected to accelerate wheat proteome and phosphoproteome studies. In the present study, a field randomized block design was used. Seeds of three biological replicates were planted and maintained under well-watered and water-deficit (rain-fed) conditions. Using TiO2 enrichment, liquid chromatography/ tandem-mass spectrometry (LC−MS/MS) analysis and labelfree phosphopeptide quantification, we performed the first comprehensive phosphoproteome analysis of the developing grains of wheat. We analyzed the Chinese bread wheat cultivar “Henong 341” under both well-watered and water-deficit conditions. Our results provide new information from the perspective of phosphoproteomics for further understanding wheat seed development and water-stressed response and defense mechanisms.



Measurement of Soil Water Content, Physiological Parameters, and Phenotypic Traits

The measurement of soil water content during different development stages, including the double-ridge stage, the jointing stage, the flowering stage, and the mature stage, followed the previously described soil drilling method of Guan et al.37 Soils from depths of 0−200 cm (counting every 20 cm as one layer) were collected, immediately placed in aluminum boxes, and dried to constant weight in an oven at 110 °C. Soil water content (%) was calculated by (C − A)/(B − A) × 100%, in which A is the weight of the aluminum box (g), B is the weight of the original soil sample and the aluminum box (g), and C is the weight of the dried soil sample and aluminum box (g). To test the effect of water-deficit and well-watered conditions on the physiological traits of flag leaves from “Henong 341” at five different time points after flowering, relative water content, proline content, and chlorophyll content were measured according to previously described methods by Larbi and Mekliche,38 Bates et al.,39 and Arnon,40 respectively. The measurement of soluble sugar and starch content was based on the procedures of Zhao et al.41 and Holm et al.42 The main phenotypic and agronomic traits of mature plants were recorded, including plant height, ear length, number of spikelets, number of infertile spikelets, kernels per spike, weight of a thousand kernels, grain length, grain width, and grain yield. Statistical analyses were conducted using independent Student’s t tests with SPSS statistics software (version 17.0). Protein Extraction

Proteins were extracted according to the procedures of Gao et al.,11 with minor modifications. Grain samples of 500 mg were ground into a meal in liquid nitrogen and then ground with 1 mL of extraction buffer containing PhosSTOP phosphataseinhibitor cocktail (1 tablet/10 mL; Roche, Basel, Switzerland), followed by placement for 3 min in extraction buffer. Samples were shaken vigorously for 15 min at room temperature. After samples were centrifuged twice, protein supernatants were precipitated with a one-quarter volume of cold 10% trichloroacetic acid at −20 °C for 2−3 h. After centrifuging, the pellets were rinsed with cold (−20 °C) acetone and then centrifuged three times at 8000 rpm for 10 min. After freeze-drying was complete, the pellets were added to 300 μL of solubilization buffer at room temperature for 2 h. After insoluble material was removed, the concentrations of protein samples were determined with a 2-D Quant Kit (Amersham Bioscience, USA), and the final protein solution was stored at −80 °C for later use. To enhance the quantitative accuracy, extracted proteins from every biological replicate were adjusted to the same concentration for the subsequent analysis.

MATERIALS AND METHODS

Wheat Materials, Field Planting, and Sampling

Chinese winter wheat cultivar “Henong 341” (Triticum aestivum L.) was grown in experimental fields of the China Agricultural University Research Center, Wuqiao, Hebei Province (116°37′23″ E, 37°16′02″ N), during the 2011−2012 growing season. Two different treatment conditions were applied at the jointing and flowering stages: well-watered (water irrigation in the field) and water-deficit (no irrigation). Each treatment included three biological replicates, and each plot was 25 m2. At the experimental location, the average annual amount of sunshine was 2690 h, and the average annual temperature was 12.6 °C. The water table ranged from 6 to 9 m, and the annual precipitation during the 2011−2012 season was 153.9 mm. The well-watered treatment at the jointing and flowering stages followed the same protocol as that applied to the cultivation of local fields: water irrigation of 750 m3/hm2 was conducted twice during the experimental period. The flag leaves of wheat at 12, 15, 18, 21, and 26 days after flowering (DAF) and the developing grains at 28 DAF from the two different treatments,

Phosphopeptide Enrichment Using TiO2 Microcolumns

Extracted protein mixtures were directly reduced with dithiothreitol (DTT), alkylated with iodoacetamide, and subsequently digested with endoproteinase Lys-C and trypsin, as described previously.43 The enrichment for the phosphopeptide procedure was performed according to Wu et al.,44 with some minor modifications. Specifically, TiO2 beads (GL Sciences, Tokyo, Japan) were incubated in 400 μL of loading buffer containing 65% acetonitrile (ACN)/2% trifluoroacetic acid (TFA) saturated with glutamic acid. Next, 2 mg of tryptic peptides was dissolved in 600 μL of loading buffer and then 4282

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Bioinformatics

incubated with the appropriate amount of TiO2 beads. After washing with 600 μL of wash buffer (65% CAN/0.1% TFA), the phosphopeptides were eluted twice with 300 μL of elution buffer (500 mM NH4OH/60% ACN). The eluates were dried and reconstituted in 0.1% formic acid (FA)/H2O for MS analysis.

The biological processes, molecular functions, and cellular components of the identified phosphoproteins were examined using Blast2GO software to perform gene ontology (GO) annotation (http://www.blast2go.com/b2ghome).46 Data sets of the protein family motif (Pfam)47 were used to identify domains that occur within proteins, a process that can provide insights into their function (http://pfam.sanger.ac.uk/). Significantly enriched phosphorylation motifs were extracted from phosphopeptides with confidently identified phosphorylation residues (class I) using the Motif-X algorithm (http://motif-x. med.harvard.edu/).29 The phosphopeptides were centered at the phosphorylated amino acid residues and aligned, and six positions upstream and downstream of the phosphorylation site were included. Because the upload restriction of Motif-X is 10 MB, a FASTA format data set (nearly 10 MB) containing the protein sequences from the wheat protein database was used as the background database to normalize the scores against the random distributions of amino acids. The occurrence threshold was set to 5% of the input data, set at a minimum of 20 peptides, and the probability threshold was set to p < 10−6. The phosphoproteins blasted by the National Center for Biotechnology Information (NCBI) were used to obtain the EuKaryotic Orthologous Groups (KOG) numbers of those proteins by eggNOG (http://eggnog.embl.de/version_3.0/). A data set containing all the KOG numbers was then used for protein−protein interaction (PPI) analysis by using the Search Tool for Retrieval of Interacting Genes/Proteins (STRING) database (http://string-db.org/). Only the phosphoproteins that had a high confidence score of at least 0.9 and were based on coexpression and experiment conditions were used to construct the network. They were then displayed using Cytoscape (version 3.0) software.48 TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) and Phyre2 (http://www.sbg.Bio.ic.ac.uk/phyre2/html/page. cgi?id=index) were used to predict the two- and threedimensional structures of certain phosphoproteins, respectively. The phosphorylated residues were displayed using Swiss-Pdb Viewer (SPDBV) software (http://spdbv.vital-it.ch/). The categories of protein kinases and phosphatases were based on PlantsP online software (plantsp.genomics.purdue.edu/).

Phosphopeptide Identification and Phosphorylation Site Localization

The enriched phosphopeptides were separated on a self-packed C18 reversed-phase column (75 μm inner diameter, 150 mm length) (Column Technology Innovation CTI, Fremont, CA) that was directly connected with a nanoelectrospray ion source in an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, America). Pump flow was split to obtain a flow rate of 1 μL/min for sample loading and 300 nL/min for the MS analysis. The mobile phases consisted of 0.1% FA (A) and 0.1% FA and 80% ACN (B). A five-step linear gradient of 5% to 30% B in 105 min, 30% to 90% B in 16 min, 90% B for 4 min, 90% to 2% B in 0.5 min, and 2% B for 14.5 min was employed. The spray voltage was set to 2.0 kV, and the temperature of the heated capillary was set at 240 °C. Each MS scan was acquired at a resolution of 60,000 (at 400 m/z), with the lock mass option enabled, and was followed by data-dependent top-10 MS/MS scans using collision-induced dissociation. The threshold for precursor ion selection was 500, and the mass window for precursor ion selection was set at 2.0 Da. The dynamic exclusion duration was 120 s, with a repeat count of 1 and repeat duration of 30 s. The analyzer used for the MS scans was Orbitrap, and the analyzer used for the MS/ MS scans was LTQ (37% relative collision energy). Three biological replicates of each sample were used independently for the phosphopeptide identification using LC−MS/MS. The raw files were processed using MaxQuant (version 1.2.2.5)45 and were compared with the wheat database. Up to two missing cleavage points were allowed. The precursor ion mass tolerance was 7 ppm, and the fragment ion mass tolerance was 0.5 Da for the MS/MS spectra. The false discovery rate (FDR) was set to p ≥ 0.5), and class III (p < 0.5), respectively (Supplemental Table S1A). Moreover, 962 (92.80%), 70 (6.80%), and 4 (0.40%) phosphorylation sites belonged to the serine, threonine, and tyrosine sites, respectively (Supplemental Figure S2B). The distribution of localization probability for the phosphorylated sites is shown in Supplemental Figure S2C. Notably, phosphorylation site probability (≥0.75) accounted for 85.60% of the total phosphorylated sites. Of the 685 identified phosphoproteins, 489 (71.38%), 118 (17.22%), 40 (5.84%), 21 (3.07%), 11 (1.61%), and 6 (0.88%) were found to have 1, 2, 3, 4, 5, and 6 or more phosphorylated sites, respectively (Supplemental Figure S2D). A total of 196 phosphoproteins contained more than two phosphorylated sites, and of these, 38 phosphoproteins had at least four phosphorylated sites. In particular, one phosphoprotein [EMT24407.1, a putative leucine repeat region (LRR) receptor-like serine/threonine-protein kinase] had 10 phos-

a

0.31 ± 0.04 0.27 ± 0.04* 39.3 ± 1.3 36.9 ± 1.4** 32.6 ± 2.2 26.4 ± 2.3* 8.9 ± 1.0 8.0 ± 1.0** 95.1 ± 2.5 90.6 ± 2.7** well-watered water-deficit

18.7 ± 2.9 17.8 ± 3.0*

3.5 ± 0.4 4.8 ± 0.4**

0.68 ± 0.05 0.57 ± 0.07*

grain width (cm) grain length (cm) WTKc (g) kernels per spike NISb spikelet numbers ear length (cm) plant height (cm) treatment

Table 1. Comparison of Agronomic Traits of Seeds in “Henong 341” under Well-Watered and Water-Deficit Conditions (n = 30)a

starch content (%)

grain yield (g/m)

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phorylated sites, and two phosphoproteins (EMT18966.1 and EMS47992.1) had 7 and 13 phosphorylated sites, respectively (Supplemental Table S1). In addition, through Pro-Q diamond and Coomassie brilliant blue (CBB) stains, some phosphoproteins of grain samples were identified under water deficit conditions (Supplemental Figure S3). Interestingly, heat shock protein 70s, phosphoglucomutases, and serpins were identified by this method. They were identified as the overlap between phosphoproteins identified through the TiO2 enrichment and PrQ stainning methods (Supplemental Table S1A and B). To increase the accuracy of the subsequent analyses, we used only phosphorylation sites from class I. All mass spectrometry proteomics data obtained in this study have been deposited in the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository50 with the data set identifier PXD000806. All mass spectrometry maps are shown in Supplemental Figure S4.

In order to obtain an overview of the phosphorylation events in the developing grains of “Henong 341” in the well-watered treatment group, the 471 identified phosphoproteins were used in GO analysis with Blast2GO software (Supplemental Table S3). GO annotation of the biological processes, molecular functions, and cellular components are shown in Figure 4. Cellular protein modification, carbohydrate metabolism, and the nucleobase-containing compound process accounted for about 50% of all phosphoproteins (Figure 4A). Another large group of phosphoproteins is involved in biotic and abiotic stimuli (14.8%). Signal transduction accounted for fewer than 7% of the phosphoproteins. Regarding the molecular functions, the three most important activities are nucleotide binding, protein binding, and kinase activity (Figure 4B). Of the total cellular components, the nucleus, plasma membrane, and cytosol accounted for 28.9%, 20.7%, and 19.8%, respectively (Figure 4C).

Phosphopeptide Screening and Function Categories under Well-Watered Conditions

Screening of Significantly Changed Phosphopeptides and Placement in Functional Categories under Water-Deficit Conditions

To increase the accuracy of phosphorylation, three biological replicates from each sample with the highest phosphorylation site localization probability (p ≥ 0.75) and phosphorylation site score differences exceeding 5 were selected. A final total of 590 phosphopeptides (containing 603 phosphorylation sites) spanning 471 phosphoproteins were identified under the well-watered treatment group (Figure 3A, Supplemental Table S2A).

To determine which phosphopeptides showed significant changes in phosphorylation status under water-deficit conditions, the intensity of each phosphopeptide was normalized to the mean of the intensities of all phosphopeptides within every biological replicate (Supplemental Table S2B). The phosphopeptides with credible Student’s t test (p < 0.05) results, significant intensity changes (≥2-fold), high phosphorylation site localization probability (≥0.75), and phosphorylation site score differences exceeding 5 were considered to exhibit significant changes in phosphorylation level (SCPL). In total, 63 SCPL unique phosphopeptides were identified, corresponding to 65 nonredundant phosphorylation sites and representing 61 phosphoproteins (Figure 3B, Supplemental Table S2B). Of note, compared with the well-watered conditions, 17 and 29 unique phosphopeptides were up regulated and down regulated (≥2-fold), respectively. A total of 4 and 14 unique phosphopeptides were specific to the well-watered and waterdeficit conditions, respectively (Table 2, Supplemental Table S2B). Interestingly, among the phosphoproteins containing at least two phosphopeptides, only one phosphopeptide showed a significant change at the phosphorylation level under waterdeficit conditions, while the other phosphopeptide showed no significant changes (Table 2). Out of these SCPL phosphoproteins, some important phosphoproteins were related to starch synthesis and protein folding/degradation (Figures 5 and 6). On the basis of KOG and Pfam query results (Supplemental Tables S4 and S5A), SCPL phosphoproteins were classified into six functional categories manually (Table 2). Of note, protein kinase and signal transduction, protein trafficking and degradation, and detoxification and defense were three important functional categories that may affect seed development and drought tolerance. Phosphorylation Motif Analysis of Developing Grains under Well-Watered or Water-Deficit Conditions

As shown in Figure 7, Motif-X results showed that only serine phosphorylation residue was enriched against total wheat protein data sets, based on accurate parameters (p < 10−6). Four different phosphorylation motifs were enriched under the well-watered conditions, [sP], [Rxxs], [sDxE], and [sxD] (Supplemental Table S6A), whereas only one motif [Rxxs] was enriched under water-deficit conditions (Supplemental Table S6B). Previous studies have indicated that these motifs were

Figure 3. Phosphorylation status in the developing grains of “Henong 341” under well-watered and water-deficit conditions. The number of phosphosites, unique phosphopeptides, and phosphoproteins that (A) showed phosphorylation status in “Henong341” under well-watered conditions and (B) exhibited significant changes in phosphorylation level under water-deficit conditions. 4286

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Figure 4. Categories of functions in the developing grains of “Henong 341” under well-watered conditions based on Blast2GO annotation: (A) biological processes, (B) molecular functions, and (C) cellular components.

also found in the seeds of Arabidopsis, rapeseed, and soybean.29 According to the published literature and databases,51−53 the motif [sP], with 195 phosphopeptides, was a proline-directed motif recognized by MAPK, cyclin-dependent kinase (CDK), and CDK-like kinase. The motif [Rxxs], with 96 phosphopeptides, was a basic motif that is a potential substrate for calmodulin kinase-II (CaMK-II), protein kinase A (PKA), and PKC. In addition, both [sxD] and [sDxE] were acidic motifs recognized by casein kinase-II (CK-II).

orthologs in all three, in two, and in only one species, respectively (Figure 8B). Only 12 (19.7%) SCPL phosphoproteins had no phosphorylated orthologs in any of the three species (Supplemental Table S7B). According to these results, the phosphoproteins of T. aestivum had the highest similarity to O. sativa, the model of monocots; relatively high similarity to A. thaliana, the model of dicots; and lower similarity to M. truncatula, the biggest phosphoprotein database presently available.

Conservation Analysis of the Phosphoproteins under Water-Deficit or Well-Watered Conditions

Category of Protein Kinases and Protein Phosphatases Identified under Well-Watered and Water-Deficit Conditions

The sequences of the phosphoproteins detected in the study were used as queries to blast the phosphoprotein databases that were constructed using data sets in the Plant Protein Phosphorylation DataBase (P3DB),54 Medicago-Omics Repository (MORE),55 and PhosPhAt 4.0.56 Medicago truncatula, Oryza sativa, and A. thaliana were compared against T. aestivum to determine the degree of conservation of phosphoproteins among different plant species. The following standards were set: Score ≥ 80, E-value < 1 × 10−10, and Identity ≥ 30%. Of the 471 phosphoproteins identified in “Henong 341”, 278 (59.0%), 81 (17.2%), and 54 (11.5%) had phosphorylated orthologs in all three, in two, and in only one species, respectively (Figure 8A). Interestingly, 58 (12.3%) phosphoproteins had no phosphorylated orthologs in any of the three species (Supplemental Table S7A). Of 61 SCPL phosphoproteins, 36 (59.0%), 8 (12.5%), and 5 (8.2%) had phosphorylated

As mentioned previously, reversible protein phosphorylation was carried out by phosphorylation of protein kinases and dephosphorylation of protein phosphatases.57 The phosphoproteins that were identified were assigned to six categories using the online PlantsP (Supplemental Table S8). Class 1 was transmembrane receptor kinases and related nontransmembrane kinases and contained seven phosphoproteins, in which three were LRR-receptor kinases (EMT09902.1, CBD32297.1, and EMT24407.1). In particular, EMT24407.1 included nine different phosphopeptides, corresponding to nine phosphorylation sites, respectively (Supplemental Figure S5), while CBD32297.1 showed high sequence similarity with other species (Supplemental Figure S6). Seven phosphorylated kinases were identified in Class 2, which contained ATN1/ CTR1/EDR1/GmPK6-like kinases. Only two casein kinase I 4287

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Table 2. Phosphoproteins with Significant Changes in Phosphorylation Level under Water-Deficit Conditions protein ID

protein descriptions

phosphosite(s)

EMT24407.1

Protein Kinase and Signal Transduction Putative LRR receptor-like serine/threonine-kinase 9

EMT10847.1 AAC49578.1 CBD32297.1 EMS55914.1 EMT18298.1 EMT14032.1 EMT23044.1

Calcium-transporting ATPase 8 Calmodulin TaCaM1-1 Unnamed protein product Hypothetical protein TRIUR3_26440 (SAP) Hypothetical protein F775_26602 Serine/threonine-protein kinase RIO1 Hypothetical protein F775_09884

Q03387.2 EMT33287.1 EMT12224.1 EMT10449.1

Transcription and Translation Factor Eukaryotic translation initiation factor isoform 4G-1 1 Eukaryotic translation initiation factor 3 subunit C 1 Nuclear transcription factor Y subunit B-4 1 Serine/arginine-rich splicing factor 4 2

EMS58509.1

Serine/arginine-rich splicing factor 7

2

EMS59731.1 EMS59675.1 EMS67637.1

Serine/arginine repetitive matrix protein 1 Serine/arginine-rich-splicing factor RSP40 Neurofilament heavy polypeptide

1 1 2

EMS47167.1 EMS52249.1 EMS67171.1

Hypothetical protein TRIUR3_03039 Hypothetical protein TRIUR3_22968 Elongation factor 2

1 1 2

EMT12320.1 EMS67868.1

Hypothetical protein F775_11049 uncharacterized protein C21orf32

1 2

EMS49802.1|

Nuclease domain-containing protein 1

1 1 1 1 1 1 2

CBH32518.1 EMS48902.1 EMS47975.1 EMS65370.1 EMS56849.1 EMT32297.1

1 Carbohydrate Metabolism Sucrose-phosphate synthase 1 ADP-glucose brittle-1 transporter precursor 1 Beta-amylase 1 Hypothetical protein F775_52380 1 Hypothetical protein TRIUR3_21203 1 Unnamed protein product 1 Acyl-CoA-binding domain-containing protein 2 1 Protein Trafficking and Degradation Ataxin-3, putative, expressed 1 DEAD-box ATP-dependent RNA helicase 40 1 Putative E3 ubiquitin-protein ligase XBOS32 1 E3 ubiquitin-protein ligase UBR4 1 14-3-3-like protein GF14-B 1 Nuclear-interacting partner of ALK 5

EMS53461.1 EMS49910.1 EMT19774.1 EMS56486.1

Hypothetical protein TRIUR3_03302 Reticulon-like protein B3 Spastin Serpin-ZX

AAR84220.1 ACX68637.1 CAA67128.1 EMT14547.1 EMS64128.1 CAJ15420.1 EMT18153.1

1 1 1 1 4288

phosphorylated peptidea _VKEEVSS(ph)PHADEAIPEK_↑ _KLDTLGDLSSQPQPVS(ph)PK_ _VSS(ph)PHAEAEMSEK_ _AADEVSS(ph)PPADVETPEK_ _ASS(ph)PDADEEIPEK_ _ATDNVSS(ph)PHADLEVPEEAADK_ _EEVSS(ph)PLADEETLEK_ _VSS(ph)PHADEATPEK_ _VSS(ph)PNADEVMPEK_ _QAALVLNAS(ph)R_↓ _DQNGFIS(ph)AAELR_↓ _NKS(ph)FDDDDDFSSKPVAR_ (+) _GRDS(ph)DDDDFR_↑ _GRDS(ph)DDEDFR_↑ _(ac)ATAAATATS(ph)PLHDSR_ _SAS(ph)DDEEEVVNTK_ (+) _ALS(ph)IDSAEGLR_ _IGDLHSES(ph)R_↑ _YTQDSDDS(ph)DTESHR_↑ _(ac)SEAVGT(ph)PESGGAK_ (+) _S(ph)PANNGSPSPGR_↑ _SPANNGSPS(ph)PGR_↑ _S(ph)ASPAPAR_↑ _SAS(ph)PAPAR_ _YAGSPLS(ph)DLEK_ (+) _ERGS(ph)PDYGR_↑ _ADS(ph)PNNMSPAANGR_ (+) _ADSPNNMS(ph)PAANGR_ (+) _GPS(ph)PLEDPALVQR_ (+) _AEGFGGES(ph)AEEK_ (+) _EQMTPLS(ph)DFEDK_↓ _FSVS(ph)PVVR_ _AGLSM(ox)S(ph)AACSWITVK_ _VQS(ph)PGLAPTAPR_ (−) _VQS(ph)PGLAPTVPR_ _LWQYGDVES(ph)DEEDQAPGGR_(+) _NFS(ph)DLSVWSDENK_↓ _LVS(ph)GAIAGAVSR_↑ _S(ph)APEELVQQVLSAGWR_↓ _S(ph)VGTLIQLQKK_↓ _ETDALTGEVQLPKS(ph)PR_↓ _AQS(ph)LPSK_↓ _LNS(ph)DVQLQLQGLLK_↓ _ERS(ph)PPSER_↑ _S(ph)RDQGPVER_↑ _EVS(ph)PVADPK_↑ _TES(ph)GDLGGSTR_↓ _EAPKNDS(ph)SEGQ_ (+) _GQESNIQHS(ph)LSCNAR_↑ _GQESNIQHSLS(ph)CNAR_ _ADSVES(ph)GEK_ _NAAS(ph)AEPDK_ _VYSGIDLS(ph)K_ _SHS(ph)NSSLNR_↑ _FHGGDSSS(ph)SSSDSDDEKK_ (+) _VVQPIISSAS(ph)DK_↓ _LASAISS(ph)PSHAK_↓ dx.doi.org/10.1021/pr500400t | J. Proteome Res. 2014, 13, 4281−4297

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Table 2. continued protein ID

protein descriptions

phosphosite(s) Protein Trafficking and Degradation 2

EMS62853.1

Spastin

EMS62287.1

Hypothetical protein TRIUR3_14133

ACV44213.1

WALI7

EMS59760.1 EMS55805.1 CAA40204.1

Disease resistance protein RPM1 DnaJ homologue subfamily C member 13 Group 3 late embryogenesis abundant protein

1 1 3

ADF31756.1

Heat shock protein 90

3

AAS68103.1 ABG68034.1 EMT14855.1

Minichromosomal maintenance factor Globulin 1 Ethylene-insensitive protein 2

1 1 2

EMS54991.1 Unknown EMS48744.1

1-Cys peroxiredoxin PER1

1

Hypothetical protein TRIUR3_08076

2

EMT12197.1 EMS66533.1 EMT33116.1

Hypothetical protein F775_18802 Hypothetical protein TRIUR3_08536 Hypothetical protein F775_32047

1 1 2

EMS63206.1

Hypothetical protein TRIUR3_14694

1

2 Detoxification and Defense 3

phosphorylated peptidea _SIS(ph)ETTLEK_↓ _NAS(ph)TSSDMSSLASQGPPSNSA _SYS(ph)LSDLR_↓ _S(ph)FTSLAEAAAR_ _INS(ph)MPR_ _QVAHAPQELNS(ph)PR_ _VDS(ph)EGVMCGANFK_↓ _S(ph)QDSFPEFVK_↓ _KSS(ph)ELNLASLAK_↓ _(ac)AS(ph)NQNQASYHAGETK_ _DQTAS(ph)TLGEK_↓ _TYETAQS(ph)AK_ _EGEVEEVDDDS(ph)ENKDESK_ _EIS(ph)DDEDEESDEKK_ _EISDDEDEES(ph)DEKK_ ↑ _S(ph)DDDGDGLTPSS(ph)PGR_↑ _GCS(ph)GESTAEQR_↑ _YHS(ph)SPDISAVIAASR_↓ _S(ph)VGTLIQLQKK_ _VATPANWKPGECVVIAPGVS(ph)DDEAK_↓ _SQS(ph)FACMSEVR_↓ _S(ph)NINLDGNQR_ _VGS(ph)AADWSSQY_↓ _TGS(ph)M(ox)AAQVK_↓ _(ac)AADGADS(ph)DLEEQLK_ (−) _EEILS(ph)EEDTTK_ _ILGEGQQNVS(ph)PR_ (−)

a

N-resize (↑) shows upregulation in phosphorylation level; S-resize (↓) shows downregulation in phosphorylation level. Plus sign (+) shows phosphorylation specifically underwater-deficit conditions; Minus sign (−) shows phosphorylation specifically under well-watered conditions.

Figure 5. Phosphorylation status of starch synthesis-related enzymes and molecular chaperones of seeds in the developing wheat grains under wellwatered and water-deficit conditions. (A) Starch synthesis pathway. (B) Protein reassembly and degradation pathway (“s” in bold type represents the sulfydryl, which can form the disulfide bond of protein through chaperone proteins). AGPase: ADP glucose pyrophosphorylase. BT1: ADPglucose brittle transporter 1. FK: fructokinase. GSP: Glucose phosphate synthetase. HSP: heat shock protein. PGI: Phosphoglucose isomerase. PGM: Phosphoglucomutase. SBE: starch branching enzyme. SS: starch synthetase. SuSy: Sucrose synthase. UGPase: UDP glucose pyrophosphorylase. The circled letter “p” represents the protein phosphorylation. Yellow circles represent phosphoproteins under the well-watered treatment, red circles indicate the phosphoproteins that showed significant changes in phosphorylation level (SCPL) under water-deficit conditions, and white circles indicate that the phosphoproteins were identified in previous studies but not in this study.

isoform delta-like proteins were phosphorylated, and they belonged to Class 3: casein kinase I (CK I). Class 4 consisted of 16 non-transmembrane protein kinases, including serine/ threonine-protein kinases PKs and Cdcs. The remaining kinases were grouped into Class 5 as other kinases. Protein phosphatases comprised Class 6. These plant phosphatases

included four, EMT22368.1, EMT12429.1, EMT32283.1, and EMT23848.1. Protein−Protein Interaction (PPI) Analysis of Protein Kinases (Phosphatase) and Other Phosphoproteins

To reveal the interactions of different protein kinases with their potential substrates, PPI analysis was conducted using STRING. Cytoscape software was used to reconstruct the 4289

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Figure 6. Sequence alignment, structural composition, three-dimensional structure, and tandem mass spectrometry (MS/MS) spectra map for Hsp90 in the developing grains of “Henong 341”. (A) Hsp90 sequence alignment with Arabidopsis, rice, and human. The red box shows the phosphorylation sites. The phosphorylation sites of other species were queried by P3DB. (B) HATPase domain, charged-linker domain, substrate binding domain, and MEEVD motif. (C)Three-dimensional structure of Hsp90 (confidence level >90%). (D) Phosphopeptide MS/MS spectra map shows the phosphorylation sites at Ser229 and Ser236.

Figure 7. Analysis of the amino acids surrounding the identified phosphorylated residues by Motif-X under well-watered conditions. “S” with blue color and “s” with red color represent phosphorylated serine residues. SP, R..S, SD.E, and SD are four different motifs enriched in our study (“.” represents any amino acid). “n” represents the number of phosphopeptide occurrences, “rs” represents the abbreviation of residue serine.

Figure 8. Conservation analysis of the “Henong 341” phosphoproteins under well-watered and water-deficit conditions. (A) Conservation analysis of phosphoproteins in the developing grains under well-watered conditions. (B) Conservation analysis of significant changes in phosphorylation level (SCPL) phosphoproteins in the developing grains under water-deficit conditions. 4290

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Figure 9. Protein−protein interaction (PPI) network of some important phosphoproteins by STRING. (A) PPI network of SAPK4 with other phosphoproteins (red node with kinases and phosphatases). (B) PPI network among a series of molecular chaperones related to protein assembly and degradation. (C) Function description of representative phosphoproteins: stress-activated protein kinase 4 (SAPK4), Ca2+/calmodulindependent protein kinase (CDPK), protein phosphatase 2C (PP2C), and heat shock protein (HSP).

signaling (AGJ93555.1) were identified in the developing grains of “Henong341”. One RLK (EMT24407.1) was shown to have nine phosphorylation sites in the two treatment conditions (Table 2). Interestingly, only the phosphopeptide at Ser1444 showed a significant increase in phosphorylation level subjected to water stress compared with well-watered conditions, while the other sites had no significant changes (Table 2). Another RLK (CBD32297.1; Supplemental Table S8) was phosphorylated at Ser381 and was present only under waterdeficit conditions. Sequence alignment analyses indicated that they were phosphorylated at the same position that corresponds to Ser381 in wheat and had a common [sxD] motif, which is a potential substrate catalyzed by CKII (Supplemental Figure S5A). Pfam and TMHMM query revealed that this phosphoprotein contained an LRR motif, a transmembrane domain, and a protein kinase catalytic domain (Supplemental Figure S5B). 3D structure analysis showed that all of these phosphorylation sites were localized in the loop region (Supplemental Figure S5C), a result that seems be in accordance with that of Lv et al.62 A recent study has shown that autophosphorylation of BR-insensitive-associated kinase 1 (BAK1), a receptor of the BR signal system, cascades BR signaling and promotes the expression of defense genes.63 In our study, the significant phosphorylation of the loop region in RLKs may facilitate its downstream substrate combination by conformational changes and play a crucial role in BR signaling cascades, thereby conferring drought tolerance during the development of wheat grain.

interaction network and display the PPI network. A total of 276 KOGs (Supplemental Table S5A and B), representing 341 phosphoproteins, were used to construct the PPI network. Using the organic layout of Cytoscape software, the most important proteins (enzymes) were enriched in the central region, whereas insignificant ones were ranked outermost. As shown in Supplemental Figure S7, 13 KOGs, representing protein kinases and phosphatases (shown with a pink outline) displayed complex interaction networks with their substrates. Interestingly, KOG0583 (SAPK4) stood out in this network for its interactions or coexpression with 53 KOG substrates (Figure 9A). Another important PPI network, representing 7 molecular chaperones, was also localized in the central position and displayed complex interactions among molecular chaperones involved in protein folding, refolding, and degradation (Figure 9B).



DISCUSSION

Protein Kinases (Phosphatases) Involved in Signal Perception and Transduction during Grain Development

Protein phosphorylation/dephosphorylation is a reversible process that is carried out by protein kinase/phosphatase in eukaryotic cells. It plays critical roles in the regulation of a large number of eukaryotic signal transductions. In this study, 43 protein kinases and four phosphatases were identified and shown to be phosphorylated (Supplemental Tables S2A and B, S8). Brassinosteroids (BRs), a signaling hormone in plants, is necessary for plant growth and development.58 Recently, many studies have reported that BR receptors that were identified in many plants are involved in embryo development and hormone signaling, as well as stress responses.59−61 In this study, two RLKs (EMT24407.1 and CBD32297.1) and a key player of BR

Transcription and Translation Factors Associated with Signaling Cascade and the Regulation of Gene Expression

After sensing and perceiving internal or external signaling, protein kinases (phosphatases) rapidly phosphorylate or dephosphorylate downstream regulatory factors, such as 4291

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revealed significant phosphorylation of BT1 under water stress, which may increase its activity and result in the transport of more starch precursor (ADPglucose) than under well-watered conditions. This indirectly explains more starch content under water stress (Table 1). Unexpectedly, phosphorylated starch synthase (SS) and starch branching enzyme (SBE) were not identified in this study. However, previous experiments in vitro have demonstrated that phosphorylated SS and SBE participate in a PPI and improve the synthesis of amylose/amylopectin.73,79,80 From these results, we speculate that to some extent these significantly phosphorylated proteins may accelerate seed ripening under water stress. Early seed ripening is known to conserve energy, which may a be a strategy for coping with adverse environments, particularly drought.

transcription and translation factors, to regulate signaling cascades. As shown in Figure 4 and Table 2, large numbers of transcription and translation factors were identified in our study and phosphorylated in both the well-watered and waterdeficit conditions (Supplemental Table S2A and B). Notably, two SR proteins (EMS58509.1 and EMS59675.1) were phosphorylated at Ser251 and Ser358, respectively, and showed a significant increase in phosphorylation level under the waterdeficit conditions. Meanwhile another SR protein (EMT10449.1) was phosphorylated at Ser285 and Ser293 and was present only under water stress conditions. Previous studies showed that a wide variety of stress signals regulated the phosphorylation status and the subcellular localization of plant SR protein in Arabidopsis.64,65 Another transcription factor, nuclear transcription factor Y subunit B-4 (NF-YB4; EMT12224.1) was phosphorylated at Thr7 and was identified only under water-deficit conditions (Supplemental Table S2). Studies have demonstrated that the NF-YB subunits, identified in maize and Arabidopsis, improve the stress-responsive physiological indexes and confer drought tolerance under water-limited conditions.66,67 In parallel, two eukaryotic translation initiation factors (Q03387.2 and EMT33287.1) were phosphorylated at Ser60 and Ser56, respectively, and showed significant phosphorylation changes compared with the well-watered conditions. Previous studies have showed that the phosphorylation of eukaryotic translation initiation factors can regulate protein synthesis and confer resistance to environmental stresses.68−70 Possibly the phosphorylation of these transcription and translation factors modifies their function in response to drought during grain development.

Chaperone and Ubiquitin Proteins Involved in Protein Refolding, Degradation, and Stress Response

Chaperone proteins act to prevent the aggregation or refolding of denatured proteins and also participate in the activation of diverse client proteins that are essential for protein folding and proteostasis.81 Our phosphoproteomics results indicated that many chaperones were phosphorylated, such as heat shock protein 90 (Hsp90), Hsp70, Hsp40, and the multifunctional chaperone14-3-3 and related proteins (Supplemental Table S2A and B). PPI analysis revealed that complex interactions occurred among these molecular chaperones (Figure 9B). Hsp90, which forms various complexes by binding different clients and co-chaperones, plays roles in the maintenance, activation, or maturation of specific client proteins in eukaryotes.82 In our study, it was identified the most and included five isomers (ADF31755.1, ADF31756.1, ADF31757.1, ABG57075.1, and ADF31772.1). As shown in Figure 6A, high sequence similarity (ADF31756.1) occurred not only in plants, including Arabidopsis and rice, but also in humans. 3D structure analyses showed that three phosphorylated sites, Ser229, Ser236, and Ser251, were distributed in the charged-linker domain, a random coiled-coil domain, whereas the three other species had the same phosphorylation site corresponding to Ser229 in wheat (Figure 6B and C). Further analysis showed that the charged-linker domain contains a large number of acidic amino acids (Figure 6A), such as Glu (E) and Asp (D), which are potential substrates catalyzed by casein kinase II.83 Notably, compared with the control, Hsp90 (ADF31756.1) showed significant increases in phosphorylation level only at Ser236 under water stress. Using deletions of the charged-linker region in Hsp90 in vivo and in vitro, a previous study demonstrated that the charged-linker region can facilitate Hsp90 interactions with the client and promote client activity by phosphorylated conformational changes.84 In addition, Tyr 24 and Thr22 in yeast and Tyr38 in human Hsp90 in vivo and in vitro have been shown to affect the ATPase activity of Hsp90 and facilitate binding to client proteins.85,86 Therefore, we suppose that the significant phosphorylation within the chargelinker region in Hsp90 may enhance its ATPase activity and indirectly promote the conjunction with client proteins. Hsp70, a key co-chaperone in the Hsp90 co-chaperone cycle,87 has a high affinity for unfolding or misfolding polypeptide chains to correct the errors. In our study it was identified and shown to be phosphorylated at Ser86 and Ser231 (EMS65063.1). Recent study indicated that C-termini phosphorylation of Hsp70 and Hsp90 facilitates binding to co-chaperones, such as CHIP (carboxyl terminus of the Hsc70-

Phosphorylated Proteins Functioning in Starch Synthesis during Grain Development

During grain development, most photosynthetic products are transported into seeds as energy materials and are involved in sucrose and starch metabolism. As shown in Figure 5A, a large number of enzymes related to starch synthesis were identified in both treatment conditions and shown to be phosphorylated, such as sucrose synthetase (SuSy, EMT12620.1, EMS65561.1), glucose-6-phosphate isomerase (GPI, EMT13737.1), ADP glucose pyrophosphorylase (AGPase, EMS55512.1), phosphoglucomutase (PGM, CAC85913.1), and ADP brittle 1 transporter (BT1, ACX68637.1) (Supplemental Table S2A and B). AGPase, the rate-limiting enzyme of starch synthesis, was phosphorylated at Ser69 under both well-watered and water-deficit conditions. It is known that AGPase, which is commonly distributed in the plastid of most plant tissues, is known to associated with the synthesis of starch.71−73 Phylogenetic analysis suggests that, in addition to being present in the plastid of Poaceae, AGPase in the cytosol of Poaceae participates in the supplemental pathway of starch synthesis in a way that is specific to the cereal endosperm.74−76 ADPglucose, a precursor of starch synthesis, is catalyzed by AGPase. The ADPglucose produced in the cytosol is transported by the BT1 transporter into plastids to participate in starch syntheses. In the current study, one ADP brittle 1 transporter (ACX68637.1) phosphorylated at Ser124 in developing grains showed a significant increase in phosphorylation level under water-deficit conditions. Large numbers of BT1 transporters have been found in the plastid envelope of crop plants, such as maize and barley.77,78 To our knowledge, there were no studies on the phosphorylation of brittle 1 transporter. Our study 4292

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proteins (CAA40204.1) was phosphorylated at Ser116 and showed significant changes in phosphorylation level under water stress, in comparison with its levels under the wellwatered conditions, while no significant changes were found for the other two sites, Ser3 and Ser102 (Table 2). A previous report showed that the phosphorylation of LEAIII protein (Rab17) in maize cells was essential to its nuclear localization, because it facilitated binding to its target proteins.98 Moreover, LEA proteins can act as osmoprotectants of nuclear or cytoplasmic macromolecules, efficiently conserving water in response to drought stress. 99 We supposed that the phosphorylation of LEA proteins could increase interactions with their substrates or determine the subcellular localization to cope with water stress. In addition, wheat aluminum induced 7 (WALI7, ACV44213.1) was phosphorylated at Ser18, Ser226, and Ser245 under both treatment conditions. However, significant phosphorylation changes occurred only at Ser226 (Table 2). Recent reports have indicated that WALI7 participates in aluminum tolerance and cell detoxification as a result of the production of ROS under water stress.100,101

interacting protein) and HOP (Hsp70−Hsp90 organizing protein), which adjusts cellular protein folding and degradation self-balancing.88 Hsp40, another co-chaperone, acts as binding to clients to form the first complex with the participation of Hsp70, which improves the association with the Hsp90 C terminus.89 In our study, five proteins containing J-domain were identified and shown to have phosphorylation modifications (EMS55805.1, EMT18416.1, EMT22588.1, EMT24229.1, and EMS60639.1). Besides the Hsp90 co-chaperone cycle, two other molecular chaperones, such as 14-3-3 proteins and 14-3-3-like proteins, are involved in the response and defense of plants under heat stress. 14-3-3 and related proteins, which are multifunctional chaperones, have become the focus of research on the stress response in plant and seed development.90−93 In our study, 143-3 protein (AGA84102.1), which was phosphorylated at Ser258, was identified in both well-watered and water-deficit conditions. Meanwhile, 14-3-3-like protein GF14-B (EMS56849.1), phosphorylated at Ser259, was present only in the water-deficit conditions. The 3D structure shows that they were both phosphorylated at the C-terminal. A previous study reported that 14-3-3 protein phosphorylated by c-Jun Nterminal kinase (Jnk) interacts with c-Abl on Thr735 and functions in nuclear targeting of c-Abl under DNA damage stress.94 In addition, Lv et al. proposed that the upregulation of 14-3-3 GF14-B phosphoprotein might bridge the ubiquitindependent pathway and participate in protein degradation under salt treatment.95 C-Terminal phosphorylation of 14-3-3 protein and GF14B may remove the inhibitory effect within the C-terminus tail and facilitate the combination with their target proteins, such as heat shock proteins, damaged proteins, and ubiquitin proteins. Generally speaking, the ubiquitin protein degradation system is controlled mainly by E3s, and most stress-related E3s act in response to environmental stress by regulating the abundance of key downstream stress-responsive transcription factors.96 Two phosphorylated E3 proteins (EMS47975.1 and EMS65370.1) showed significant changes in phosphorylation level under water stress, compared with well-watered conditions. It was reported that PKA-dependent phosphorylation of the E3 ligase could convert its substrate preference, contributing to selective protein degradation.97 During water stress, the specific binding and interaction of phosphorylated Hsp90 and co-chaperones might enhance their activities and facilitate the conformational maturation of client proteins. Through the Hsp90-co-chaperone cycle and ubiquitin-dependent pathway, mismatched and unfolding proteins of clients were refolded to natural proteins, while denatured and damaged proteins were selected for degradation (Figure 5B). This might be a protective strategy to protect cells from damage induced by a variety of stresses, particularly drought.

An Overview of Stress Response and Defense Mechanisms during Grain Development in Wheat

When subjected to drought stress, developing grains first perceive the external stimulus. Some signal molecules, such as ABA and BR, are produced in cells. Subsequently, each signal molecule activates its corresponding protein kinase. Activated protein kinase phosphorylates its downstream transcription and translation factor(s) to facilitate the binding to specific genes and then induce the production of stress-related proteins. To cope with drought stress, these proteins are also phosphorylated by specific protein kinase(s). Some of these phosphorylated proteins participate in osmotic protection and ROS scavenging, while others participate in water and sugar transport to regulate osmotic balance.



CONCLUSIONS

This study was the first comprehensive phosphoproteome analysis of developing grains in wheat under well-watered and water-deficit conditions. A total of 471 phosphoproteins were detected in vivo using label-free quantification. Through comparative phosphoproteome analysis, 61 phosphoproteins showed significant changes under water stress. Function analysis suggested that some SCPL phosphoproteins are involved in signal transduction, the synthesis of starch, protein refolding/degradation, and stress response and defense. According to previous studies, protein phosphorylation occurs in three ways: (1) through an increase in the activity itself, (2) through the binding to specific substrates by conformational changes, and (3) through the determination of subcellular localization. Through these phosphorylation mechanisms, some SCPL proteins may play important roles in the signal transduction and signaling cascade of grain development under water stress. To cope with water deficit, these SCPL proteins may function in early ripening of grains, the repair and scavenging of damaged proteins, osmotic regulation, and cellular detoxification.

Phosphoproteins Associated with the Cellular Stress Response, Detoxification, and Defense

During the late stages of seed development and maturity, large numbers of hydrophobic proteins gradually accumulate to protect cells from adverse environments, including biotic and abiotic stresses. Several hydrophobic proteins identified under both conditions were found to be phosphorylated in this study, including two late embryogenesis abundant (LEA) proteins (CAA40204.1 and AAN74639.1), one dehydrin (DHN; EMT30993.1), and two early methionine (EM) proteins (P42755.1 and P04568.1). Interestingly, one of the LEA 4293

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ASSOCIATED CONTENT

S Supporting Information *

Supplemental tables and figures as described in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Tel and Fax: +86-10-68902777. E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research was financially supported by grants from the National Natural Science Foundation of China (31271703, 31101145), International Science & Technology Cooperation Program of China (2013DFG30530), Natural Science Foundation of Beijing City and the Key Developmental Project of Science and Technology, Beijing Municipal Commission of Education (KZ201410028031), and the National Key Project for Transgenic Crops in China (2011ZX08009-003-004).



ABBREVIATIONS AGPase, ADP glucose pyrophosphorylase; BAK1, BR insensitive-associated kinase 1; BR, brassinosteroid; CID, collision induced dissociation; DAF, days after flowering; FDR, false discovery rate; GO, gene ontology; KOG, eukaryotic orthologous group; LEA, late embryogenesis abundant proteins; MAPK, mitogen-activated protein kinase; MORE, Medicago-Omics Repository; NCBI, National Center for Biotechnology Information; P3DB, phosphorylation protein database; Pfam, protein family motif; PP2C, protein phosphatase 2C; PPI, protein−protein interaction; RLK, receptor-like kinase; SAPK4, stress-associated protein kinase 4; SCPL, significant changes in phosphorylation level; SnRK, snf1-related kinase; STRING, search tool for retrieval of interacting genes/proteins; WALI7, wheat aluminum induced 7



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