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

Abstract

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.

Keywords: Cold Stress; Mass Spectrometry; Phosphoproteome; Phosphorylation; Signal Transduction; Stress response; Tomato Phosphoproteome.

Fig. 1.

A universal sample preparation protocol for plant phosphoproteomics. A, Schematic representation of the comparison of plant lysis buffers, contaminant removal strategies, and tryptic digestion buffers to analyze tomato phosphoproteomics using 200 μg of tomato leaf protein. B, Comparison of the plant tissue lysis efficiency using Tris-HCl, SDC and SLS mixture, and GdnHCl lysis protocols. C, Venn diagram showing the overlap of the number of identified phosphopeptides from triplicate analyses of the three lysis protocols. D, Evaluation of the performance of FASP and methanol-chloroform precipitation for improving phosphopeptide identification. Comparison of (E) the phosphoproteomic coverage and (F) the percentage of missed cleavage between the urea and SDC-SLS tryptic digestion protocols.

Fig. 2.

Profiling the phosphoproteome of tomato leaves in response to cold stress. A, The tomatoes leaves were grown in three biological replicates for 5 days at room or cold temperature. Proteins were extracted, precipitated, and digested using the optimized protocol. The digested phosphopeptides were dimethyl-labeled, combined, and enriched using PolyMAC. To enlarge the phosphoproteome coverage, the isolated phosphopeptides were fractionated using basic pH reverse-phase StageTips prior to LC-MS/MS analysis. B, The total number of identified phosphoproteins, phosphopeptides, and phosphorylation sites in the phosphoproteome of tomato leaves. C, Distribution of the number of phosphates and amino acid residues for all detected phosphopeptides.

Fig. 3.

Phosphoproteomic and kinome perturbation because of cold response in tomatoes. A, Principle component analysis (PCA) of the localized phosphorylation sites across all four samples representing distinct phosphoproteomic types for different treatments. B, Unsupervised hierarchical clustering of significantly changed phosphorylation sites of all biological replicates under cold stress (ANOVA, permutation-based FDR < 0.05). The color code of each phosphorylation site (row) in all samples (columns) indicates the low (blue) and high (red) Z-score normalized intensities. C, Phosphorylation motif analysis of the phosphorylation sites induced in Atacames and N135 Green Gage in response to cold stress. D, Cold stress regulates the phosphorylation sites on tomato kinases. Ranked list of phosphorylation changes of kinases in response to cold treatment. Dots show the mean fold change of three biological replicates.

Fig. 4.

Identifying the direct substrates of SnRK2E involved in cold responses in S. lycopersicum. A, The KALIP workflow was applied to identify the direct SnRK2E substrates in N135 Green Gage with high confidence. B, Examples of enriched phosphorylation motifs from the identified heavy phosphorylation sites of in vitro SnRK2E screening. C, The overlap of in vitro kinase screening and in vivo phosphoproteomic results to acquire the candidate substrates of SnRK2E. D, Schematic representation of the proposed cold-induced mechanisms through activation of SnRK2E in N135 Green Gage. The bar graph shows the cold-induced changes in phosphorylation levels of proteins between the two tomatoes.