Comparative phosphoproteome analysis upon ethylene and abscisic acid treatment in Glycine max leaves

Plant Physiology and Biochemistry 130 (2018) 173–180

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Research article

Comparative phosphoproteome analysis upon ethylene and abscisic acid treatment in Glycine max leaves

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Ravi Guptaa, Cheol Woo Mina, Qingfeng Menga, Ganesh Kumar Agrawalb,c, Randeep Rakwalc,d,e, Sun Tae Kima,∗ a

Department of Plant Bioscience, Life and Industry Convergence Research Institute, Pusan National University, Miryang, 627-707, Republic of Korea Research Laboratory for Biotechnology and Biochemistry (RLABB), GPO Box 13265, Kathmandu, Nepal c GRADE Academy Private Limited, Adarsh Nagar-13, Birgunj, Nepal d Faculty of Health and Sport Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8574, Japan e Global Research Center for Innovative Life Science, Peptide Drug Innovation, School of Pharmacy and Pharmaceutical Sciences, Hoshi University, 4-41 Ebara 2-chome, Shinagawa, Tokyo, 142-8501, Japan b

A R T I C LE I N FO

A B S T R A C T

Keywords: ABA Ethylene Phosphoproteins Soybean Transporters Kinase

Abscisic acid (ABA) and ethylene play key roles in growth and development of plants. Several attempts have been made to investigate the ABA and ethylene-induced signaling in plants, however, the involvement of phosphorylation and dephosphorylation in fine-tuning of the induced response has not been investigated much. Here, a phosphoproteomic analysis was carried out to identify the phosphoproteins in response to ABA, ethylene (ET) and combined ABA + ET treatments in soybean leaves. Phosphoproteome analysis led to the identification of 802 phosphopeptides, representing 422 unique protein groups. A comparative analysis led to the identification of 40 phosphosites that significantly changed in response to given hormone treatments. Functional annotation of the identified phosphoproteins showed that these were majorly involved in nucleic acid binding, signaling, transport and stress response. Localization prediction showed that 67% of the identified phosphoproteins were nuclear, indicating their potential involvement in gene regulation. Taken together, these results provide an overview of the ABA, ET and combined ABA + ET signaling in soybean leaves at phosphoproteome level.

1. Introduction Abscisic acid (ABA) and ethylene (ET) are involved in the regulation of diverse biological pathways including normal growth, development and stress response (Bari and Jones, 2009; Müller and Munné-Bosch, 2015; Salazar et al., 2015; Sharp, 2002). Growing body of evidence suggests a potential cross-talk between ABA and ET signaling during several biological processes including seed dormancy, germination and fruit development (Arc et al., 2013; Weng et al., 2015; Zhang et al., 2009). In most of the cases, if not all, ABA and ET work in an antagonistic manner (Arc et al., 2013). It was reported that exogenous application of ET precursor counteracts the inhibitory effects of ABA on endosperm cap weakening and endosperm rupture in Arabidopsis and Lepidium sativum (Arc et al., 2013; Linkies et al., 2009). ABA limits the ET production by inhibiting the ACC oxidase (ACO) activity and reduces ACO transcription, a major enzyme involved in ET biosynthesis (Arc et al., 2013). Moreover, recent reports have shown that the exogenous application of ethephon (ET precursor) leads to the

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accumulation of dietary isoflavones in soybean leaves and the effect was partially inhibited by the application of ABA (Gupta et al., 2018a; Yuk et al., 2016). Because of the antagonistic effects of these two hormones, our previous study investigated the effect of ABA, ET and combined ABA + ET on soybean leaves using a high-throughput proteomics approach. This study highlighted the involvement of MAP kinase in the regulation of these hormone signaling. In particular, phosphorylation of MAPK3, MAPK4, and MAPK6 was observed in response to ET treatment while ABA treatment led to the dephosphorylation of these MAP kinases. These results indicate a potential involvement of phosphorylation and dephosphorylation in the regulation of these hormone induced signaling in soybean leaves. Phosphorylation is a well-known post–translational modification that regulates the activity of proteins involved in the myriad of biological pathways (Kline-Jonakin et al., 2011). Phosphorylation and dephosphorylation of proteins by kinases and phosphatases respectively, act as a biological switch that regulates the functional properties of proteins (Kline-Jonakin et al., 2011). Attempts have been made

Corresponding author. Department of Plant Bioscience, Pusan National University, Miryang, 627-706, South Korea. E-mail address: [email protected] (S.T. Kim).

https://doi.org/10.1016/j.plaphy.2018.07.002 Received 20 April 2018; Received in revised form 2 July 2018; Accepted 2 July 2018 Available online 04 July 2018 0981-9428/ © 2018 Elsevier Masson SAS. All rights reserved.

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2.3. Phosphopeptide identification

previously to identify the phosphoproteins in response to ABA or ET treatments (Bhaskara et al., 2017; Li et al., 2009; Minkoff et al., 2015; Qing et al., 2016; Qiu et al., 2017; Umezawa et al., 2013; Wang et al., 2013). A recent study where phosphoproteomic changes in grapevine leaves were analyzed after ABA treatment identified 33 differential proteins using a label-free quantitative proteomic approach (Rattanakan et al., 2016). Of these differential proteins, abundance of 20 and 13 phosphoproteins was increased and decreased respectively, upon ABA treatment. Proteins related to the serine family amino acid metabolic process showed decreased abundance while protein folding related proteins were upregulated in response to ABA treatment (Rattanakan et al., 2016). Similarly, several other studies led to the identification of hundreds of phosphopeptides in response to ABA or ET treatments, however, none of these studies focused on investigating the combined effect of ABA and ET. Moreover, there is no report of either ABA or ET phosphoproteome analysis in soybean. Therefore, here we carried out a phosphoproteome analysis to analyze the changes in soybean (Glycine max) leaf upon ET, ABA, and combined ABA + ET treatments. Results presented here provide new insights into the molecular action of ET and ABA signaling and their consequences on the metabolic pathways in soybean leaves.

Enriched phosphopeptides were desalted using zip-tips, dissolved in solvent-A (water/ACN, 98:2 v/v; 0.1% formic acid), and separated by reversed-phase chromatography using a UHPLC Dionex UltiMate® 3000 (Thermo Fisher Scientific, USA) instrument (Gupta et al., 2016). For trapping the sample, the UHPLC was equipped with Acclaim PepMap 100 trap column (100 μm × 2 cm, nanoViper C18, 5 μm, 100 Å) and subsequently washed with 98% solvent A for 6 min at a flow rate of 6 μL/min. The sample was continuously separated on an Acclaim PepMap 100 capillary column (75 μm × 15 cm, nanoViper C18, 3 μm, 100 Å) at a flow rate of 400 nL/min. The LC analytical gradient was run at 2%–35% solvent B over 90 min, then 35%–95% over 10 min, followed by 90% solvent B for 5 min, and finally 5% solvent B for 15 min. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was coupled with an electrospray ionization source to the quadrupole-based mass spectrometer QExactive™ Orbitrap High-Resolution Mass Spectrometer (Thermo Fisher Scientific, USA). Resulting peptides were electro-sprayed through a coated silica emitted tip (PicoTip emitter, New Objective, USA) at an ion spray voltage of 2000 eV. The MS spectra were acquired at a resolution of 70,000 (200 m/z) in a mass range of 350–1800 m/z. A maximum injection time was set to 100 ms for ion accumulation. Eluted samples were used for MS/MS events (resolution of 17,500), measured in a data-dependent mode for the 10 most abundant peaks (Top10 method), in the high mass accuracy Orbitrap after ion activation/dissociation with Higher Energy C-trap Dissociation (HCD) at 27 collision energy in a 100–1650 m/z mass range (Min et al., 2017).

2. Materials and methods 2.1. Plant growth and hormone treatments Soybean seeds (Glycine max cv. Daewon) were planted in the soil and allowed to grow in a growth chamber at 25 °C (16/8 h day/light cycle, 70% relative humidity) for one month. Ethephon (5 mM, a natural precursor of ET) and ABA (500 μM) were prepared in the deionized water and 50 mL of solution was evenly sprayed, using a foliar spray, over six pots containing five soybean plants per pot. An equal volume of deionized water was sprayed as a control. Whole trays were shifted to transparent acrylic chambers and sealed to prevent the evaporation of ET produced. Leaves were harvested after 3 h for phosphoproteome analysis.

2.4. Data processing using MaxQuant software The obtained raw data were analyzed by MaxQuant software (Cox and Mann, 2008) v.1.5.0.0 using Andromeda as a search engine (Cox et al., 2011). The acquired MS/MS spectra were searched against the soybean protein database (Gmax_275_Wm82. a2. v1, 88647 entries), obtained from Phytozome and quantification of peptides and proteins was performed by MaxQuant with an FDR < 0.01 for proteins, peptides, and modifications. Search parameters included trypsin as a cleavage enzyme, cysteine carbamidomethylation as a fixed modification and oxidation of methionine, acetylation (protein N-term), and phosphorylation of Ser, Thr, Tyr residue (phosphoSTY) as variable modifications. A minimum peptide length of six amino acids was specified and “match between runs” (MBR) was enabled with a matching time window of 1 min. Obtained data were analyzed using Perseus software and phosphopeptides that were reproducibly identified in at least two out of three replicates of at least one sample with score > 40 and delta score > 7 were considered as valid identification and used for the further analysis.

2.2. Protein extraction and phosphopeptide enrichment Control and hormone-treated leaves (1 g) were homogenized in 5 mL RIPA buffer containing phosSTOP phosphatase inhibitor cocktail (Roche, Basel, Switzerland) and protease inhibitor cocktail (Thermo Fisher Scientific, USA) and subjected to methanol-chloroform precipitation. The pellets so obtained were solubilized in 1 × SDS-loading buffer or 6 M urea for SDS-PAGE or in-solution trypsin digestion respectively. Protein concentration in each fraction was determined by 2D-Quant Kit (GE Healthcare, Uppsala, Sweden) and 30 μg protein from each fraction was loaded on 12% SDS-PAGE. Gels were either stained with the PRO-Q diamond stain (Invitrogen, OR, USA) as per the manufacturer's instructions or electroblotted on PVDF membranes and probed with antiphospho-serine (Q5) or antiphospho-threonine (Q7) antibodies (Qiagen SJ, USA). For shotgun proteome analysis, 1 mg protein from each sample was used for in-solution trypsin digestion as described previously (Gupta et al., 2016). In brief, proteins were first reduced using 1 mM dithiothreitol (DTT) and then alkylated with 5.5 mM iodoacetamide for 45 min. Urea concentration was reduced to 0.6 M by the addition of deionized water and digested overnight with Trypsin Gold (Promega, Madison, USA). Protein digest was acidified to a final concentration of 0.1% TFA to stop the trypsin digestion. Peptides were centrifuged at 2000 g for 10 min to remove the undigested proteins and desalted using C18 columns (Empore, MN, USA) prior to phosphopeptide enrichment. TiO2 based phosphopeptide enrichment kit (Pierce Biotechnology) was used for enrichment of phosphopeptides following manufacturer's protocol.

2.5. Functional annotation of the identified proteins 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/ ). The phosphopeptides were centered at the phosphorylated amino acid residues and aligned, and six positions upstream and downstream of the phosphorylation site were included. A data set containing the Arabidopsis homologs of the identified phosphoproteins were retrieved from phytozome and used for protein−protein interaction (PPI) analysis by the Search Tool for Retrieval of Interacting Genes/Proteins (STRING) database (http://string-db.org/) and arranged using Cytoscape. Functional analysis of the identified proteins was carried out using DAVID functional annotation tool (https://david.ncifcrf.gov/ tools.jsp) with integrated PANTHER Gene Ontology (GO) and KEGG pathways. 174

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Fig. 1. (A) SDS-PAGE profile showing phosphoproteins stained by PRO-Q diamond phosphoprotein stain. Western blots of the same samples probed against anti phospho-serine (B) and anti phospho-threonine (C) antibodies. (D) CBB stained SDS-PAGE gel of the same samples showing equal amount (30 μg) of protein loaded in each well. Arrows indicate differentially modulated phosphoproteins.

Fig. 2. (A) Venn diagram showing the distribution of phosphopeptides in different sample sets. (B) Pie chart showing number of phosphosites in the identified phosphopeptides. (C) Localization prediction of the identified phosphoproteins using CELLO-subcellular localization predictive system. (D) Identification of enriched phosphorylation motifs in the identified phosphoproteins using Motif-x program. Highly enriched motifs around phosphoserine and phosphothreonine sites. (E) Table showing all the identified motifs with their scores and fold increase.

3. Results

more treatments (Fig. 1A). In particular, a polypeptide of 100 kDa was found to be specifically phosphorylated in response to ABA treatment (Fig. 1A). In addition to the PRO-Q diamond staining, changes in the phosphoproteome were also checked by the Western blots using phospho-serine and phosphor-threonine antibodies. Western blots with phospho -serine and -threonine antibodies showed a distinct phosphoprotein profile with some phosphoproteins specifically identified with phosphoserine antibodies while others with phosphothreonine (Fig. 1B and C). A close look at the membranes showed dephosphorylation of 18 kDa, 20 kDa, 23 kDa, 37 kDa polypeptides upon ABA treatment (Fig. 1B) while an increase in the phosphorylation of 23 kDa and 25 kDa

3.1. Gel-based screening of phytohormones induced phosphoproteome changes At first, total proteins isolated from control and hormones treated soybean leaves were resolved on SDS-PAGE and stained by PRO-Q diamond phosphoprotein fluorescence stain. Several phosphoproteins ranging from 10 to 150 kDa were observed on the PRO-Q diamond stained SDS-PAGE gel of which polypeptides at 110 kDa, 39 kDa, 30 kDa and 21 kDa appeared to be phosphorylated in response to one or 175

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Fig. 3. (A) Functional annotation of the identified phosphoproteins using PANTHER GO. (B) MapMan analysis highlighting “biotic stress overview”. Phosphosite intensities of the identified phosphopeptides are shown by red-blue color scale. (C) Protein-protein interaction network of the phosphoproteins involved in ABA signaling, oxidative stress, photosynthesis, protein autophosphorylation and protein transport using Cytoscape integrated with STRING. Abbreviations: C-control, AABA, E-ethylene, AE-ABA + ET. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

identified phosphopeptides showed that 63% of these contained one phosphorylation site, 36% contained two and only 1% contained three phosphorylation sites (Fig. 2B). Subcellular localization prediction showed that 66% of the identified phosphopeptides were localized in the nucleus with only 14% proteins predicted as cytoplasmic and 9% as chloroplastic (Fig. 2C). Moreover, 9%, 1%, and 1% phosphoproteins were predicted to be plasma membrane, mitochondria and Golgi localized (Fig. 2C). As majority of the identified phosphoproteins were nuclear, it clearly indicates their potential involvement in gene regulation. Moreover, identification of chloroplast and plasma membrane localized proteins indicate a vast regulation of photosynthesis and transport related proteins by phosphorylation in response to ABA and ET treatments.

polypeptides was observed in response to ET treatment (Fig. 1C). These results indicate that phytohormone treatment results in differential phosphorylation of some of the proteins and are worth investigating (Fig. 1D).

3.2. Shotgun proteomics approach for the identification of phytohormone responsive phosphoproteins Following the gel-based approach, a shotgun proteomics approach was utilized to identify the phosphoproteome changes upon phytohormones treatment in soybean leaves. Phosphopeptides were enriched using TiO2 and were identified by the QExactive high-resolution mass spectrometer. This approach led to the identification of a total of 802 phosphopeptides matching with 422 protein groups. To increase the reliability and accuracy, phosphopeptides that were identified in at least two of the three replicates of at least one sample were selected and used for further analysis. Using this cutoff, a total of 686 reproducible phosphopeptides were identified of which 592 showed a localization probability ≥0.75, score > 40 and a delta score of > 7 and were considered as class I phosphosites (see supplementary table 1 in reference Gupta et al., 2018b). The rest of the sites fell into class II and class III categories as per the classification given previously; yet the probability that these peptides are phosphorylated is still larger than 99% (Olsen et al., 2006). Of these identified phosphopeptides, 499 (78%) were common to all the sample sets, 2 were specific to ET and 13 were specific to the ABA + ET treatment (Fig. 2A). Further analysis of the

3.3. Identification of kinase motifs in the enriched phosphopeptides In order to identify the kinases involved in the phosphorylation of the identified phosphoproteins, the conserved motifs around the identified phosphosites were screened. PhosphoSitePlus database identified substrate motifs for casein kinase II (27.92%), 14-3-3 domain binding motif (19.27%), b-adrenergic receptor kinase (10.31%), casein kinase I (9.86%), ERK1,2 kinase (8.19%), calmodulin-dependent protein kinase (5.15%), G protein-coupled receptor kinase 1 (4.24), PKA kinase (2.12) and MAPKAPK2 kinase (1.06%) (see supplementary table 1 in reference Gupta et al., 2018b). Moreover, overpresented motifs around the phosphosites were also identified by an online Motif-X tool (http:// 176

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Fig. 4. Bar diagrams showing phosphosite intensities of some of the significantly modulated phosphosites in response to phytohormone treatments. Phosphorylated amino acid with their phosphosite localization probability is highlighted by the red color at the bottom of each graph. Abbreviations: C-control, A-ABA, E-ethylene, AE-ABA + ET. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

biotic stress-responsive phosphoproteins were further identified by MapMan analysis using phosphosite intensities. Results showed a general increase in the phosphorylation of PR-proteins in response to all the treatments (Fig. 3B). Heat shock proteins were majorly increased in response to ABA and ET treatments while these showed a dephosphorylation when ABA and ET were present together. ABA signaling related proteins were phosphorylated in response to ABA and ABA + ET treatment while these were dephosphorylated in response to ET treatment and same was found to be true for WRKY transcription factors. In case of cell wall and proteolysis related proteins, an overall increase in phosphorylation was observed in case of ABA + ET treatment. Moreover, a general increase in the phosphorylation was observed in case of signaling related proteins in response to all the treatments. In particular, increased MAPK phosphorylation was observed in response to ET and ABA + ET treatment (Fig. 3B). A protein-protein interaction network was constructed to get an insight into the functional interaction of the identified phosphoproteins. Using STRING database combined with Cytoscape, a total of 1206 experimentally confirmed interactions were observed among the identified phosphoproteins (see Fig. 1 in reference Gupta et al., 2018b). Of these, proteins specifically involved in the ABA signaling, oxidative stress, photosynthesis, protein autophosphorylation and protein transport, as per the PANTHER functional class, were selected and again used for the development of interactome. Of the total 99 selected proteins for the analysis, 524 interactions were observed with an average of 5 interactions per protein (Fig. 3C). These results indicate that the identified phosphoproteins function in a highly coordinated manner to perform the biological response.

motif-x.med.harvard.edu/motif-x.html) using a 13 amino acid sequence, centered by S, T, and Y phosphorylation sites. Using Arabidopsis PhosPho site database as background data, a total of 16 motifs were extracted from our data of which 13 were centered to phosphoserine sites while 3 were centered around phosphothreonine sites (Fig. 2D and E). In the case of phosphoserine, [sxS] was the most abundant motif with 213 matches, followed by [sP] and [Sxs] while [Sxxxxxt] was identified as the most abundant phosphothreonine motif (Fig. 2E).

3.4. Functional annotation of the identified phosphoproteins DAVID functional annotation tool with integrated PANTHER protein class and gene ontology (GO) databases was used to decipher the functions of the identified phosphoproteins. In the “cellular component” category of GO-annotation tool, phosphoproteins related to cell part (45%), organelle (29%), macromolecular complex (19%) and membrane (5%) were highly enriched (Fig. 3A). In case of “molecular function” category, phosphoproteins related to the catalytic activity (46%), binding (37%), structural molecular activity (7%), transporter activity (7%) and antioxidant activity (3%) were highly enriched (Fig. 3A). Biological process category showed enrichment of metabolic process (35%), cellular process (33%), cellular component organization (8%), response to stimulus (7%), biological regulation (7%) and localization (7%). PANTHER protein class analysis showed that phosphoproteins with nucleic acid binding activity (23%) were the major class followed by hydrolase (14%), oxidoreductase (12%), transferase (7%) and transporter (7%). Moreover, 4% of the identified phosphoproteins were transcription factors, indicating their involvement in gene regulation (Fig. 3A). These results indicate that the identified phosphoproteins were involved in the various cellular processes and thus could regulate almost all the major pathways of plant metabolism. Both ABA and ET have a role in biotic stress tolerance, therefore,

3.5. Identification of phytohormone induced differential phosphosites It is well known that phosphorylation and dephosphorylation of the proteins act as a biological switch that regulates the functional 177

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potential substrates for ERK1/2 kinase activity and interestingly 94.44% of these contained class I phosphosites (see supplementary table 1 in reference Gupta et al., 2018b). In addition, we identified 1.06% of the phosphopeptides, as potential targets of the MAPKAPK2 kinase, supporting the role of MAPK-signaling in ET and ABA signaling pathways. Furthermore, identification of a MAPKKK7 (Glyma.11G101700.3. p) as phosphorylated protein further supports a central role of MAPK signaling pathway in the ET signaling pathway. MAPKs are the members of serine/threonine protein kinases, involved in the fine-tuning of several biological processes including plant defense and induction of ET biosynthesis (Meng and Zhang, 2013). Using a Western blotting approach, a previous study confirmed the involvement of MAPK-signaling in ET signaling in soybean leaves (Gupta et al., 2018a).

properties of the proteins. Therefore, in order to identify the ABA, ET and combined ABA + ET induced changes in phosphorylation, multiple sample test was applied on the 686 identified phosphopeptides. A total of 40 phosphopeptides showing significant differences (p < 0.05) in the phosphosite intensity were identified (see supplementary table 1 in reference Gupta et al., 2018b, Fig. 4). These differential phosphopeptides were majorly nucleus (50%), plasma membrane (20%) and cytoplasm (15%) localized (see supplementary table 1 in reference Gupta et al., 2018b) and were majorly belong to hydrolase (16%), nucleic acid binding (16%), transporter (16%), membrane traffic protein (11%), oxidoreductase (11%) and transferase (11%) classes, as per the PANTHER-protein class classification (see Fig. 2 in reference Gupta et al., 2018b). 4. Discussion

4.1. ABA signaling phosphorylate several plasma membrane localized transporters

Both ABA and ET play crucial roles in the physiology of plants. However, and to date, many of these phytohormones-regulated physiological processes remain poorly understood, especially at the phosphoproteome level. Holistic understanding of hormone signaling and their global functions is of paramount biological importance to understand the physiology of plants during normal growth and stress conditions. Therefore, to identify the ABA and ET-induced signaling components modulated by phosphorylation, this study investigated the changes in the phosphoproteome using a shotgun proteomics approach. Gel-based detection of phosphoproteins using PRO-Q diamond staining and Western blotting showed an almost similar profile of phosphoproteins. However, some high-molecular weight phosphoproteins at 100 kDa and 110 kDa were only observed in the PRO-Q diamond stained gel while some low-molecular weight phosphoproteins such as 18 kDa and 20 kDa phosphoproteins, were only detected in the Western blots, suggesting different selectivity of these two phosphoproteins detection systems. In addition to phospho-serine and phosphothreonine, PRO-Q diamond stain reacts with phospho-tyrosine moieties also and thus it is possible that the high-molecular weight phosphoproteins, detected only in the PRO-Q diamond stained gel, are phosphorylated at tyrosine moieties, however, this needs further investigations. Using shotgun proteomics approach, this study identified 686 phosphopeptides and 40 differential phosphosites upon phytohormone treatments of which 66% were found to be nucleus localized. Similar results have been reported previously where the abundance of nuclear protein phosphorylation was observed in rice seedlings in response to ABA treatment (Qiu et al., 2017) and bacterial blight infection (Hou et al., 2015). Nuclear proteins are involved in a plethora of biological functions including gene regulation and DNA replication, among others and thus phosphorylation of nuclear proteins could affect multiple biological processes. Kinase motif analysis using Motif-X tool led to the identification of 16 conserved kinase motifs of which [sxS] was most prominent while [sP] was most enriched. [sxS] motif has been previously identified in rice in response to bacterial blight infection and Arabidopsis, however, its functional kinases are still unknown. [sP] is one of the most commonly identified motifs in plants including Arabidopsis, rice and wheat and is the target motif for many kinases including MAPK, SnRK2 (sucrose non-fermenting1-related protein kinase 2), RLK (receptor-like kinase), AGC (cAMP-dependent, cGMP-dependent and protein kinase C), CDK (cyclin-dependent kinase), CDPK (calcium-dependent protein kinase) and SLK (STE20-like kinase) (Hou et al., 2015; van Wijk et al., 2014). In the case of phospho-threonine motifs, [tP] is most common (van Wijk et al., 2014); however, it was not identified in our study. Here, we identified [Sxxxxxt] as the most abundant phosphothreonine motif which is a target motif for MAPKK6 (MKK6) (Xie et al., 2012). ERK1 and ERK2 are the effector kinases of the ERK1/2 MAPK signaling pathway which also participate in hormone signaling (Frémin et al., 2015). Our results showed 8.19% of total phosphopeptides as

Localization prediction showed 9% of the identified phosphoproteins to be plasma membrane localized. Functional annotation of these proteins showed that majority of these were transporters including plasma membrane intrinsic proteins (PIPs, Glyma.14G061500.3. p, Glyma.13G325900.1. p, Glyma.20G179700.1. p, Glyma.12G172500.1. p), transmembrane amino acid transporter family protein (TATP, Glyma.01G161100.1. p, Glyma.11G082700.1. p), K+ uptake permease 7 (Glyma.08G072500.1. p), ammonium transporter 1; 2 (Glyma.10G168100.1. p), K+ efflux antiporter 2 (Glyma.09G262000.1. p), sodium hydrogen exchanger 2 (Glyma.10G158700.1. p, Glyma.20G229900.1. p), autoinhibited Ca2+ -ATPase isoform 8 (Glyma.09G061200.5. p), pleiotropic drug resistance 7 (Glyma.08G201300.2. p), ABC-2 type transporter family protein (Glyma.12G020400.3. p), and ABC subfamily B1 (Glyma.13G142100.2. p) and B19 (Glyma.13G063700.2. p). Of these, a TATP (Glyma.11G082700.1. p), phosphorylated at tyrosine residue, showed increased phosphorylation in response to combined ABA + ET treatment. TATPs are involved in the transportation of amino acids across the membranes and availability of amino acids modulates the gene expression and signal transduction pathways. There are reports which indicate that the altered concentrations of amino acids across the membranes trigger a signaling cascade that modulates the gene expression and/or protein activity (Forde and Roberts, 2014). Thus, differential phosphorylation of TATP upon combined ABA + ET treatment suggest that the concentration of amino acid regulates, at least partially, the effect of these two hormones when present together. PIPs are aquaporins which are primarily involved in the transportation of water molecules across the membrane. Moreover, growing body of evidence suggests their potential roles in both biotic and abiotic stress tolerance (Jang et al., 2004; Lee et al., 2012; Tian et al., 2016). It was observed that the transcript levels of many PIPs modulate differentially in response to drought, cold, salinity, or ABA treatment in Arabidopsis (Jang et al., 2004) with maximum change observed after drought stress and minimum by salinity stress. Phosphorylation of PIPs has been shown previously during temperature dependent opening of tulip petals and their phosphorylation was predicted to be catalyzed by CDPK (Azad et al., 2004). Here, we identified six phosphopeptides corresponding to the PIPs and were predicted to be the substrate(s) for the casein kinase I, casein kinase II, PKA kinase or PKC kinase with no prediction of CDPK (see supplementary table 1 in reference Gupta et al., 2018b). Therefore, further experimental evidences are required to identify the exact kinase responsible for the phosphorylation of PIPs. Moreover, it is also possible that different PIPs require different kinases to get phosphorylated and thus needs further investigations. ABC transporters are involved in multiple functions including transport of ABA (Kang et al., 2011, 2010). Identification of plasma membrane-localized ABC transporters indicate that these must be involved in the intake of ABA from outside to inside the cells and 178

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locus receptor kinase (SRK) to be activated and carry out the proteasomal degradation of the target proteins to reject the self-incompatible pollen in Brassica (Stone et al., 2003). Meanwhile, the ABA and ET concentrations were also found to be higher in the incompatible flowers, indicating a potential role of both these hormones in the regulation of self-incompatibility (Baker et al., 1997). Here, we observed the increased phosphorylation of ARM-repeat superfamily protein in response to ABA treatment and based on the previous findings, it can be expected that the ABA induces SRK which in turns phosphorylates ARM-proteins in presence of ABA. Ribosomal protein S6e, a part of 40S ribosomal subunit, is involved in the regulation of translation and its phosphorylation is well-known in both plants and animals. It was reported that the translation of stored mRNA in germinating maize axes is mediated by the phosphorylation of S6 ribosomal protein (Sánchez-De-Jiménez et al., 1997). Further studies indicated the role of auxin in inducing the phosphorylation of this particular protein and thus in the regulation of protein synthesis in maize axes (Beltrán-Peña et al., 2002). Here, increased phosphorylation of ribosomal proteins S6e was observed, particularly in response to ET treatment, suggesting that ET also regulate the protein synthesis by modulating the phosphorylation induced function of this particular protein in soybean leaves. PTEN (phosphatase and tensin homologue deleted on chromosome ten) proteins exhibit phosphatase activity and utilize protein and phosphoinositides as substrates (Pribat et al., 2012). Moreover, it was also shown that the PTEN2a interacts with phosphatidic acid, a wellknown second messenger in plants. Furthermore, phosphoinositides, targets of PTEN proteins, are inositol containing phospholipids that are involved in multiple functions including signal transduction and stomatal movements (Lee and Lee, 2008). Here, we observed decreased phosphorylation of PTEN2 in response to ET treatment and increased phosphorylation in response to combined ET + ABA treatment, indicating that the ET and combined ET + ABA signaling modulates the lipid signaling by turning on/off the biological activity of PTEN proteins by phosphorylation. DUF1645 protein showed increased phosphorylation in response to ET and ABA + ET treatment. These proteins are involved in multiple functions including response to biotic as well as abiotic stress. It was shown that overexpression of a DUF1645, STRESS_tolerance and GRAIN_LENGTH (OsSGL), in rice increases its overall yield by increasing the grain length, grain weight and grain number per panicle (Wang et al., 2016) and drought tolerance (Cui et al., 2016). Further analysis of OsSGL-overexpressing plants indicates a role of cytokinin signaling, however, ET and ABA + ET induced modulation of DUF1645 by phosphorylation suggests an additional level of regulation of this protein by ET and combined ABA + ET signaling in soybean.

phosphorylation of these proteins play a distinct role in this transportation. Pleiotropic drug resistance (PDR) proteins are the members of ABC transporters that are only found in fungi and plants (Crouzet et al., 2006). These proteins are involved in the transportation of antimicrobial compounds to the cell surface and thus play a role in the plant defense (Crouzet et al., 2006). In our study, we found increased phosphorylation of two phosphopeptides corresponding to the PDR proteins upon combined ABA + ET treatment, indicating the role of phosphorylated PDR proteins in the plant defense triggered by these two hormones. Similarly, identification of other transporters indicates a central role of phosphorylation in regulation of cellular transport. It is well known that ABA signaling modulates several ion channels and transporters, therefore, identification of these proteins as phosphorylated proteins further indicate that ABA-induced modulation of these proteins might be mediated by the phosphorylation and dephosphorylation of these proteins. 4.2. ET and ABA induced differential phosphorylation of transcription factors and nuclear proteins A significant proportion (67%) of the identified phosphoproteins were found to be nucleus localized and PANTHER protein class analysis also showed that the identified phosphoproteins were involved in the nucleic acid binding activity (23%) and transcription factors (4%), among others. Among the significantly modulated proteins, 18 were nuclear including RGPR-related, plant protein 1589 of unknown function, J-domain protein required for chloroplast accumulation response 1 (JAC1), tetratricopeptide repeat (TPR)-like superfamily protein, CCCH-type zinc finger family protein, protein of unknown function (DUF1645), kinase-related protein of unknown function (DUF1296), ARM repeat superfamily protein, PTEN 2, phototropin 1 (phot1), phototropin 2 (phot2), WUS-interacting protein 2, serine-rich protein-related, ribosomal protein S6e and an unknown protein. Although, RGPR-related proteins are relatively understudied in plants, its phosphorylation has been already reported. Phosphorylated RGPR binds the TTGC motifs in the promoter regions of several genes to alter their expression (Yamaguchi, 2009). In this study, a ABA induced dephosphorylation of RGPR was observed, suggesting that ABA negative regulates the expression of RGPR induced genes. JAC1 and phototropins (phot1 and phot 2) are involved in the phototropic responses in plants. Phototropins are blue light receptors that contain Ser/Thr kinase domain at their C-terminus and gets autophosphorylated when exposed to blue light (Sakai et al., 2001). During low light conditions, chloroplasts accumulate towards the light, which is known as accumulation response and JAC1 is a positive regulator of accumulation response in Arabidopsis (Suetsugu et al., 2005). Besides, phototropism, phototropins are also involved in the stomatal opening by activating and/or phosphorylating H+-ATPase of the guard cells (Kinoshita et al., 2001; Sakai et al., 2001). Involvement of ABA in stomatal movement is well established and thus the involvement of ABA in regulating the phototropin activity can be expected. However, a recent study reported that the mRNA levels of both phot 1 and phot 2 were independent of ABA in light with a slight downregulation of phot1 by ABA in dark. Moreover, protein levels of both of these phototropins were downregulated by the ABA (Eckstein et al., 2016). These results suggest a negative regulation of phototropins by ABA. Here, we observed a dephosphorylation of both of these phototropins in response to ABA, suggesting that in addition to regulating the protein levels, ABA also regulate the activity of these proteins at the post-translational level. Phototropins are involved in the opening of stomata while ABA is involved in the closing of stomata, thus it is possible that the ABA induced closure of stomata is mediated by the inhibition of photoropins both at translational and post-translational levels. ARM repeat superfamily proteins are involved in multiple functions including abiotic stress signaling. An ARM repeat-containing protein-1 (ARC1) is an E3-Ubiquitin ligase and needs to be phosphorylated by S-

5. Conclusion This study utilized a shotgun proteomics approach to identify the ABA, ET and combined ABA + ET responsive phosphoproteins in soybean leaves. This approach led to the identification of a total of 686 phosphopeptides of which 40 showed a significant change in phosphorylation in response to one or more phytohormone treatments. Results obtained here suggest that the ABA regulates multiple plasma membrane-localized transporters and ion channels by phosphorylation. Furthermore, several other proteins identified and discussed indicate the phosphorylation-mediated regulation of various cellular processes including stress response, stomatal movements and protein synthesis, among others. Author contributions RG conceived and designed the study, conducted the research, and wrote the paper; STK was involved in the study design; CWM and MQF helped RG in data analysis; RR and GKA supplemented the writing. All 179

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authors have read and approved the final manuscript.

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