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Universal Plant Phosphoproteomics Workflow and Its Application to Tomato Signaling in Response to Cold Stress*

  • Chuan-Chih Hsu
    Footnotes
    Affiliations
    Department of Biochemistry, Purdue University, West Lafayette, IN 47907
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  • Yingfang Zhu
    Footnotes
    Affiliations
    Department of Horticulture and Landscape, Purdue University, West Lafayette, IN 47907

    Shanghai Center for Plant Stress Biology and Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China

    Institute of Plant Stress Biology, State Key Laboratory of Cotton Biology, Department of Biology, Henan University, Kaifeng 475001, China
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  • Justine V. Arrington
    Affiliations
    Department of Chemistry, Purdue University, West Lafayette, IN 47907
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  • Juan Sebastian Paez
    Affiliations
    Department of Biochemistry, Purdue University, West Lafayette, IN 47907
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  • Pengcheng Wang
    Affiliations
    Institute of Plant Stress Biology, State Key Laboratory of Cotton Biology, Department of Biology, Henan University, Kaifeng 475001, China

    Shanghai Center for Plant Stress Biology and Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China
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  • Peipei Zhu
    Affiliations
    Department of Chemistry, Purdue University, West Lafayette, IN 47907
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  • I-Hsuan Chen
    Affiliations
    Department of Biochemistry, Purdue University, West Lafayette, IN 47907
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  • Jian-Kang Zhu
    Affiliations
    Department of Biochemistry, Purdue University, West Lafayette, IN 47907

    Institute of Plant Stress Biology, State Key Laboratory of Cotton Biology, Department of Biology, Henan University, Kaifeng 475001, China

    Shanghai Center for Plant Stress Biology and Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China
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  • W. Andy Tao
    Correspondence
    To whom correspondence should be addressed:Department of Biochemistry, Purdue University, West Lafayette, IN 47907.
    Affiliations
    Department of Biochemistry, Purdue University, West Lafayette, IN 47907

    Department of Chemistry, Purdue University, West Lafayette, IN 47907
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  • Author Footnotes
    §§ The authors contributed to the project equally.
    This article contains supplemental material. The authors have declared no conflict of interest.
Open AccessPublished:July 13, 2018DOI:https://doi.org/10.1074/mcp.TIR118.000702
      Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against environmental stresses. To address the high complexity and dynamic range of plant proteomes and phosphoproteomes, we present a universal sample preparation procedure that facilitates plant phosphoproteomic profiling. This advanced workflow significantly improves phosphopeptide identifications, enabling deep insight into plant phosphoproteomes. We then applied the workflow to study the phosphorylation events involved in tomato cold tolerance mechanisms. Phosphoproteomic changes of two tomato species (N135 Green Gage and Atacames) with distinct cold tolerance phenotypes were profiled under cold stress. In total, we identified more than 30,000 unique phosphopeptides from tomato leaves, representing about 5500 phosphoproteins, thereby creating the largest tomato phosphoproteomic resource to date. The data, along with the validation through in vitro kinase reactions, allowed us to identify kinases involved in cold tolerant signaling and discover distinctive kinase-substrate events in two tomato species in response to a cold environment. The activation of SnRK2s and their direct substrates may assist N135 Green Gage tomatoes in surviving long-term cold stress. Taken together, the streamlined approach and the resulting deep phosphoproteomic analyses revealed a global view of tomato cold-induced signaling mechanisms.
      Protein phosphorylation is crucial for plant cells in perceiving and responding to environmental stimuli through transduction of signals from receptor kinases to targets (
      • Zhu J.K.
      Abiotic Stress Signaling and Responses in Plants.
      ,
      • Schulze W.X.
      Proteomics approaches to understand protein phosphorylation in pathway modulation.
      ). Compared with those of humans, plant kinases are double in number and diversity, highlighting the importance of the plant kinome and phosphoproteome in regulating responses to both abiotic and biotic stresses (
      • Silva-Sanchez C.
      • Li H.
      • Chen S.
      Recent advances and challenges in plant phosphoproteomics.
      ,
      • Singh D.K.
      • Calvino M.
      • Brauer E.K.
      • Fernandez-Pozo N.
      • Strickler S.
      • Yalamanchili R.
      • Suzuki H.
      • Aoki K.
      • Shibata D.
      • Stratmann J.W.
      • Popescu G.V.
      • Mueller L.A.
      • Popescu S.C.
      The tomato kinome and the tomato kinase library ORFeome: novel resources for the study of kinases and signal transduction in tomato and solanaceae species.
      ). Therefore, profiling the phosphoproteomic changes in response to environmental stresses is an efficient way to understand and to delineate a global view of plant defense mechanisms. Cold stress is a major environmental factor that affects the growth, distribution, and yield of many important crops growing in tropical or subtropical areas (
      • Chinnusamy V.
      • Zhu J.
      • Zhu J.K.
      Cold stress regulation of gene expression in plants.
      ,
      • Miura K.
      • Furumoto T.
      Cold signaling and cold response in plants.
      ,
      • Thomashow M.F.
      Plant Cold Acclimation: Freezing Tolerance Genes and Regulatory Mechanisms.
      ). Under prolonged cold temperatures, plant cells alter the expression of thousands of genes to reach a cold acclimation status. Many important transcription factors are expressed under cold stress to regulate plant cold acclimation (
      • Chinnusamy V.
      • Zhu J.
      • Zhu J.K.
      Cold stress regulation of gene expression in plants.
      ,
      • Medina J.
      • Catala R.
      • Salinas J.
      The CBFs: three arabidopsis transcription factors to cold acclimate.
      ). For example, the increase of transcription factor ICE1 expression plays a role in the modulation of cold-responsive genes (CORs) such as the expression of three CBF genes in Arabidopsis (
      • Chinnusamy V.
      • Ohta M.
      • Kanrar S.
      • Lee B.H.
      • Hong X.
      • Agarwal M.
      • Zhu J.K.
      ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.
      ). Under cold stress, ice1 mutant plants had reduced expression of CBF genes and displayed the cold sensitive phenotype (
      • Chinnusamy V.
      • Ohta M.
      • Kanrar S.
      • Lee B.H.
      • Hong X.
      • Agarwal M.
      • Zhu J.K.
      ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.
      ). Besides the important role of transcriptional factors in cold acclimation, many plant kinases are activated and positively regulate plant freezing tolerance at the post-translational level. One of the canonical events is the activation of MEKK1-MKK2-MAPK4/6 cascades in Arabidopsis under short periods of cold treatment, which has been linked to enhanced freezing tolerance (
      • Teige M.
      • Scheikl E.
      • Eulgem T.
      • Doczi R.
      • Ichimura K.
      • Shinozaki K.
      • Dangl J.L.
      • Hirt H.
      The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis.
      ,
      • Furuya T.
      • Matsuoka D.
      • Nanmori T.
      Phosphorylation of Arabidopsis thaliana MEKK1 via Ca(2+) signaling as a part of the cold stress response.
      ). For example, a phosphoproteomics study revealed that the phosphorylation level of Thr31 on MKK2 was highly increased in the cold-tolerant banana (Musa app. Dajiao) but not in the cold-sensitive Cavendish banana (
      • Gao J.
      • Zhang S.
      • He W.D.
      • Shao X.H.
      • Li C.Y.
      • Wei Y.R.
      • Deng G.M.
      • Kuang R.B.
      • Hu C.H.
      • Yi G.J.
      • Yang Q.S.
      Comparative phosphoproteomics reveals an important role of MKK2 in banana (Musa spp.) cold signal network.
      ). Another example is the serine/threonine kinase OST1, one of the core components of the Abscisic acid (ABA)
      The abbreviations used are:
      ABA
      abscisic acid
      CAA
      2-chloroacetamide
      CKL2
      casein kinase 1 like protein 2
      FASP
      filter aided sample preparation
      GdnHCl
      guanidine hydrochloride
      MAPK
      mitogen activated protein kinase
      MWCO
      molecular weight cut-off filter
      PolyMAC
      polymer-based metal-ion affinity capture
      PTS
      phase-transfer surfactant
      SDC
      sodium deoxycholate
      siKALIP
      stable isotope labeling kinase assay linked phosphoproteomics
      SLS
      sodium lauroyl sarcosinate
      SnRK2
      sucrose non-fermenting 1-related protein kinase 2
      TCEP
      Tris(2-carboxyethyl)phosphine hydrochloride
      TSAP
      temperature-sensitive alkaline phosphatase.
      1The abbreviations used are:ABA
      abscisic acid
      CAA
      2-chloroacetamide
      CKL2
      casein kinase 1 like protein 2
      FASP
      filter aided sample preparation
      GdnHCl
      guanidine hydrochloride
      MAPK
      mitogen activated protein kinase
      MWCO
      molecular weight cut-off filter
      PolyMAC
      polymer-based metal-ion affinity capture
      PTS
      phase-transfer surfactant
      SDC
      sodium deoxycholate
      siKALIP
      stable isotope labeling kinase assay linked phosphoproteomics
      SLS
      sodium lauroyl sarcosinate
      SnRK2
      sucrose non-fermenting 1-related protein kinase 2
      TCEP
      Tris(2-carboxyethyl)phosphine hydrochloride
      TSAP
      temperature-sensitive alkaline phosphatase.
      pathway, which modulates ICE1 protein turnover through phosphorylation at Ser278 (
      • Ding Y.
      • Li H.
      • Zhang X.
      • Xie Q.
      • Gong Z.
      • Yang S.
      OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis.
      ). Phosphorylation of this site prevents the degradation of ICE1 protein under cold stress, which promotes cold tolerance in Arabidopsis. These examples suggest that the plant phosphoproteome and kinome are involved in the regulation of molecular events that trigger cold acclimation.
      As the tomato is one of the most important horticultural crops in the world, we utilized our proteomic approach to investigate the underlying mechanisms of their cold tolerance. Considering the limited knowledge of the molecular mechanisms that regulate tomato cold tolerance, different tomato subtypes with distinct cold tolerances are suitable as a model system to study these mechanisms. Previous reports have systematically compared the cold tolerances of tomatoes in the view of the transcriptome (
      • Chen H.
      • Chen X.
      • Chen D.
      • Li J.
      • Zhang Y.
      • Wang A.
      A comparison of the low temperature transcriptomes of two tomato genotypes that differ in freezing tolerance: Solanum lycopersicum and Solanum habrochaites.
      ,
      • Patade V.Y.
      • Khatri D.
      • Kumari M.
      • Grover A.
      • Mohan Gupta S.
      • Ahmed Z.
      Cold tolerance in Osmotin transgenic tomato (Solanum lycopersicum L.) is associated with modulation in transcript abundance of stress responsive genes.
      ). Distinct gene expression patterns were observed from different cold tolerant tomato variants, indicating the complexity of cold-induced molecular mechanisms in tomatoes. However, there has been no large-scale study to characterize the roles of the tomato kinome and phosphoproteome in the regulation of cold tolerance. We recently observed that a novel cultivated tomato, N135 Green Gage (cultivar, Solanum lycopersicum), is tolerant of prolonged cold exposure (4 °C), whereas a wild tomato, Atacames (PIM, Solanum. pimpinellifolium), displays a cold-sensitive phenotype under cold conditions. Thus, these tomatoes are ideal materials to study the important cold tolerance signaling pathways in tomato.
      Mass spectrometry (MS) has emerged as a powerful technology for identifying thousands of phosphorylation sites in a single shot from mammalian cells and extracellular vesicles (
      • Imamura H.
      • Sugiyama N.
      • Wakabayashi M.
      • Ishihama Y.
      Large-scale identification of phosphorylation sites for profiling protein kinase selectivity.
      ,
      • Tsai C.F.
      • Hsu C.C.
      • Hung J.N.
      • Wang Y.T.
      • Choong W.K.
      • Zeng M.Y.
      • Lin P.Y.
      • Hong R.W.
      • Sung T.Y.
      • Chen Y.J.
      Sequential phosphoproteomic enrichment through complementary metal-directed immobilized metal ion affinity chromatography.
      ,
      • Humphrey S.J.
      • Azimifar S.B.
      • Mann M.
      High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics.
      ,
      • Chen I.H.
      • Xue L.
      • Hsu C.C.
      • Paez J.S.
      • Pan L.
      • Andaluz H.
      • Wendt M.K.
      • Iliuk A.B.
      • Zhu J.K.
      • Tao W.A.
      Phosphoproteins in extracellular vesicles as candidate markers for breast cancer.
      ). However, significant analytical difficulty is encountered in plant phosphoproteomics because of the high dynamic range of the plant proteome, the rigidity of plant cell walls, and the interference from chlorophyll and secondary metabolites (
      • Isaacson T.
      • Damasceno C.M.
      • Saravanan R.S.
      • He Y.
      • Catala C.
      • Saladie M.
      • Rose J.K.
      Sample extraction techniques for enhanced proteomic analysis of plant tissues.
      ,
      • Wang W.
      • Tai F.
      • Chen S.
      Optimizing protein extraction from plant tissues for enhanced proteomics analysis.
      ,
      • Kim Y.J.
      • Lee H.M.
      • Wang Y.
      • Wu J.
      • Kim S.G.
      • Kang K.Y.
      • Park K.H.
      • Kim Y.C.
      • Choi I.S.
      • Agrawal G.K.
      • Rakwal R.
      • Kim S.T.
      Depletion of abundant plant RuBisCO protein using the protamine sulfate precipitation method.
      ). These challenges hamper the sensitivity and efficiency of detecting low abundance phosphorylation events in plants through MS. In addition, resources concerning the tomato phosphoproteome are still limited by the lack of a full genomic sequence of the tomato. Stulemeijer et al. only reported 50 phosphoproteins from tomato seedlings using TiO2 enrichment and LC-MS/MS analysis, which was partially attributed to poor spectrum and protein sequence matching (
      • Stulemeijer I.J.
      • Joosten M.H.
      • Jensen O.N.
      Quantitative phosphoproteomics of tomato mounting a hypersensitive response reveals a swift suppression of photosynthetic activity and a differential role for hsp90 isoforms.
      ). To address the limits of global plant phosphoproteomics, we have carefully evaluated the performance of several techniques that have been previously employed in each proteomic sample preparation step. We introduce here a universal sample preparation protocol, which significantly increased the coverage and depth of the plant phosphoproteome. This protocol was then applied to study the phosphoproteomic perturbation of two tomato varieties under prolonged cold treatment. This in-depth phosphoproteomic resource reveals the phosphorylation sites implicated in kinase activation and cold-responsive gene expression. Upon coupling this data with in vitro kinase screening, we discovered a connection between SnRK2s activation and cold tolerance through phosphorylation of their downstream kinases, which sheds light upon which tomato phosphoproteins are critical for conferring cold tolerance.

      DISCUSSION

      The high complexity and dynamic range of the plant proteome is a major challenge in studying plant phosphoproteomics. We achieved efficient coverage of the plant phosphoproteome by enhancing the lysis efficiency, removing the interfering species, and optimizing the digestion protocols, which permitted identification of many low abundant phosphorylation sites from both cytosol and organelles without depletion of high abundance proteins such as RuBisCo prior to PolyMAC enrichment. This optimized protocol is applicable to studies of global phosphoproteomic remodeling of any given plant species after environmental or genetic perturbation. We have demonstrated that more than 14,000 class I phosphorylation sites corresponding to 5000 phosphoproteins can be identified in two novel tomato species that display distinct tolerance phenotypes after prolonged cold stress, which provides an informative resource to the community to study cold acclimation mechanisms in tomatoes.
      More importantly, this work not only provides the deepest quantitative analysis of the tomato cold-induced phosphoproteome to date but also partially reveals the underlying mechanisms conferring the cold tolerance in tomatoes at the post-translational level. Through phosphorylation motif analysis, we identified known molecular mechanisms in cold-treated Atacames (S. pimpinellifolium) and further discovered new kinase-substrate relationships that may regulate cold tolerance in N135 Green Gage (S. lycopersicum). This analysis shows that the two tomato varieties have distinct kinome remodeling during prolonged cold stress. For example, several MAPKs are activated in Atacames in response to a cold environment, whereas in N135 Green Gage two SnRK2 kinases are activated. The data is supported by a previous report that the concentration of ABA is significantly increased in S. lycopersicum after 12 h and continues rising until 72 h under exposure to cold temperature (10 °C) (
      • Daie J.
      • Campbell W.F.
      Response of tomato plants to stressful temperatures: increase in abscisic acid concentrations.
      ). Because ABA is the upstream activator of the kinase SnRK2E, we propose that the induction of SnRK2E activity is attributed to the high production of ABA in N135 Green Gage under prolonged cold stress.
      The overrepresented acidic phosphorylation motif in cold-treated N135 Green Gage suggests that kinases recognizing the acidic motif maybe involved in cold acclimation signaling in N135 Green Gage. It has been reported that the expression of CKL2 is induced during drought stress and ABA treatment, and CKL2 participates in stomatal closure to prevent water loss by phosphorylating and physically interacting with actin depolymerizing factor 4 (ADF4) to prevent its degradation (
      • Zhao S.
      • Jiang Y.
      • Zhao Y.
      • Huang S.
      • Yuan M.
      • Zhao Y.
      • Guo Y.
      CASEIN KINASE1-LIKE PROTEIN2 regulates actin filament stability and stomatal closure via phosphorylation of actin depolymerizing factor.
      ). Considering that phosphorylation levels on the casein kinases increase after cold stimulation in N135 Green Gage, it is possible that the casein kinases are activated in this species to alleviate water loss caused by prolonged cold exposure. Furthermore, casein kinases are known to recognize acidic phosphorylation motifs, and the motif [-pS-d-] is significantly enriched in the clusters of phosphorylation sites associated with cold-treated N135 Green Gage (clusters 4 and 5). The combination of our phosphoproteomic data with in vitro SnRK2E kinase-substrate screening enabled us to link the cold-induced acidic ([-pS-d-]) motif with the activation of SnRK2E in N135 Green Gage. Our results suggest that SnRK2E phosphorylates CKL2 under cold stress in N135 Green Gage, and CKL2 further phosphorylates many downstream substrates with an acidic motif to regulate cold tolerance. We also observed that SnRK2E directly phosphorylates several sites of ATM and histone H2A in N135 Green Gage, suggesting a potentially important role for the SRK-ATM-H2A axis in N135 Green Gage cold stress.
      In summary, we have developed a novel phosphoproteomic pipeline for profiling the tomato phosphoproteome and have illustrated its application to the study of signal transduction in tomatoes under cold exposure. More importantly, we characterized the induction of different kinases in two tomato varieties which show distinct cold phenotypes. Our analyses identified novel tomato signaling cascades that regulate cold acclimation. Together, we expect that this large-scale quantitative phosphoproteomic data will serve as a useful database to aid in identification of key enzymes that trigger cold tolerance in tomatoes.

      DATA AVAILABILITY

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (52) via the jPOST partner repository (53) with the dataset identifier PXD010310.

      Acknowledgments

      We are grateful for financial support from the NSF (grant 1506752).

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