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The recent discovery of SPINDLY (SPY)-catalyzed protein O-fucosylation revealed a novel mechanism for regulating nucleocytoplasmic protein functions in plants. Genetic evidence indicates the important roles of SPY in diverse developmental and physiological processes. However, the upstream signal controlling SPY activity and the downstream substrate proteins O-fucosylated by SPY remain largely unknown. Here, we demonstrated that SPY mediates sugar-dependent growth in Arabidopsis (Arabidopsis thaliana). We further identified hundreds of O-fucosylated proteins using lectin affinity chromatography followed by mass spectrometry. All the O-fucosylation events quantified in our proteomic analyses were undetectable or dramatically decreased in the spy mutants, and thus likely catalyzed by SPY. The O-fucosylome includes mostly nuclear and cytosolic proteins. Many O-fucosylated proteins function in essential cellular processes, phytohormone signaling, and developmental programs, consistent with the genetic functions of SPY. The O-fucosylome also includes many proteins modified by O-linked N-acetylglucosamine (O-GlcNAc) and by phosphorylation downstream of the target of rapamycin (TOR) kinase, revealing the convergence of these nutrient signaling pathways on key regulatory functions such as post-transcriptional/translational regulation and phytohormone responses. Our study identified numerous targets of SPY/O-fucosylation and potential nodes of crosstalk among sugar/nutrient signaling pathways, enabling future dissection of the signaling network that mediates sugar regulation of plant growth and development.

Ischemic diseases including myocardial infarction (MI) and limb ischemia are some of the greatest causes of morbidity and mortality worldwide. Cell therapy is a potential treatment but is usually limited by poor survival and retention of donor cells injected at the target site. Since much of the therapeutic effects occur via cell-secreted paracrine factors, including extracellular vesicles (EVs), we developed a porous material for cell encapsulation which would improve donor cell retention and survival, while allowing EV secretion. Human donor cardiac mesenchymal cells were used as a model therapeutic cell and the encapsulation system could sustain three-dimensional cell growth and secretion of therapeutic factors. Secretion of EVs and protective growth factors were increased by encapsulation, and secreted EVs had hypoxia-protective, pro-angiogenic activities in in vitro assays. In a mouse model of limb ischemia the implant improved angiogenesis and blood flow, and in an MI model the system preserved ejection fraction %. In both instances, the encapsulation system greatly extended donor cell retention and survival compared to directly injected cells. This system represents a promising therapy for ischemic diseases and could be adapted for treatment of other diseases in the future.

Glioblastoma multiforme (GBM), which is a malignant primary brain tumor, is the cancer that spreads most aggressively into the adjacent brain tissue. Patients with metastatic GBM have a poor chance of survival. In this study, we examined the anti-GBM mobility effect of small protein, called GMI, which is cloned and purified from Ganoderma microsporum. Proteomic profiles showed that GMI-mediated proteins were involved in cell motility and cell growth functions. Specifically, we demonstrated that GMI significantly suppressed cell migration and invasion of GBM cells. GMI combined with temozolomide (TMZ), which is a traditional chemotherapeutic agent for GBM treatment, synergistically inhibited motility in GBM cells. Mechanistically, we demonstrated that GMI induced proteasome-dependent degradation of Slug, which is a critical transcription factor, is frequently linked to metastasis and drug resistance in GBM. Knockdown of Slug reduced cell viability and colony formation of GBM cells but enhanced TMZ-suppressed cell migration and viability. The results of this study show that targeting Slug degradation is involved in GMI-suppressed mobility of GBM cells. Moreover, GMI may be a potential supplementary agent for the suppression of GBM.

Abiotic stresses have significant impacts on crop yield and quality. Even though significant efforts during the past decade have been devoted to uncovering the core signaling pathways associated with the phytohormone abscisic acid (ABA) and abiotic stress in plants, abiotic stress signaling mechanisms in most crops remain largely unclear. The core components of the ABA signaling pathway, including early events in the osmotic stress-induced phosphorylation network, have recently been elucidated in Arabidopsis with the aid of phosphoproteomics technologies. We now know that SNF1-related kinases 2 (SnRK2s) are not only inhibited by the clade A type 2C protein phosphatases (PP2Cs) through dephosphorylation, but also phosphorylated and activated by upstream mitogen-activated protein kinase kinase kinases (MAP3Ks). Through describing the course of studies to elucidate abiotic stress and ABA signaling, we will discuss how we can take advantage of the latest innovations in mass-spectrometry-based phosphoproteomics and structural proteomics to boost our investigation of plant regulation and responses to ABA and abiotic stress.

Supplementary Figure Legend

Gene list for Fig.5B, C, D

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle–regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651–66. ©2017 AACR.

Supplemental data on additional models to support the role of EZH2 in RTKi resistance

Diversification of effector function, driven by a co-evolutionary arms race, enables pathogens to establish compatible interactions with their hosts. Structurally conserved plant pathogenesis-related PR-1 and PR-1-like (PR-1L) proteins are involved in plant defense and fungal virulence, respectively. It is unclear how fungal PR-1L counteracts plant defense. Here, we show that Ustilago maydis UmPR-1La and yeast ScPRY1 with conserved phenolic detoxification functions are Ser/Thr-rich region-mediated cell-surface localization proteins. However, UmPR-1La has gained additional specialized activity in eliciting hyphal-like formation, suggesting that U. maydis deploys UmPR-1La to sense phenolics and direct their growth in plants. U. maydis also hijacks plant cathepsin B-like 3 (CatB3) to release functional CAPE-like peptides after cleaving a conserved CNYD motif of UmPR-1La to subvert plant immunity for promoting fungal virulence. Surprisingly, CatB3 avoids cleavage of plant PR-1s, despite the presence of the same conserved CNYD motif. Our work highlights that UmPR-1La has acquired additional dual roles to suppress plant defense and sustain the infection process of fungal pathogens.

Mass spectrometry-based proteomics provide a powerful tool for plant research, allowing global detection of steady-state levels of proteins under a given experimental setup. Here, we provide an optimized protocol for proteomic profiling using tandem mass tag (TMT) labeling followed by liquid chromatography-mass spectrometry (LC-MS/MS) to quantitate phosphopeptides and non-phosphopeptides from the same samples. The outlined protocol comprises a series of successive steps, namely, SDS (sodium dodecyl sulfate) protein extraction, protein precipitation, digestion, TMT labeling, phosphopeptide enrichment, high pH reversed-phase fractionation, LC-MS/MS analysis, protein identification, and data analysis. Our proteome-scale protocol requires 0.1 mg protein per sample and allows for the reliable and accurate quantification of more than 8000 proteins in Arabidopsis plant samples across multiple conditions, including low abundant peptides.

Protein phosphatases play an essential role in normal cell physiology and the development of diseases such as cancer. The innate challenges associated with studying protein phosphatases have limited our understanding of their substrates, molecular mechanisms, and unique functions within highly coordinated networks. Here, we introduce a novel strategy using substrate-trapping mutants coupled with quantitative proteomics methods to identify physiological substrates of Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) in a high-throughput manner. The technique integrates three parallel mass spectrometry-based proteomics experiments, including affinity isolation of substrate-trapping mutant complex using wild-type and SHP2 KO cells,iin vivo/iglobal quantitative phosphoproteomics, andiin vitro/iphosphatase reaction. We confidently identified 18 direct substrates of SHP2 in the epidermal growth factor receptor signaling pathways, including both known and novel SHP2 substrates. Docking protein 1 was further validated using biochemical assays as a novel SHP2 substrate, providing a mechanism for SHP2-mediated Ras activation. This advanced workflow improves the systemic identification of direct substrates of protein phosphatases, facilitating our understanding of the equally important roles of protein phosphatases in cellular signaling.

This study conducted a systematic review following the guidelines of the PRISMA statement, with the aim of analyzing the scientific production on Education for Sustainable Development (ESD) in in-service and pre-service mathematics teachers. Out of 211 articles identified in the indexed databases, WOS, SCOPUS, and SciELO, 16 were selected for an exhaustive analysis. It was determined that this topic is still in the process of development, focusing on aspects such as learning content, pedagogy, and the learning environment. Most studies employed qualitative methodologies. Contextualization in sustainability is the most advanced competency. The results highlight the importance of continuing to investigate and develop effective strategies to integrate ESD in the continuous education and training of mathematics teachers. It is essential to address the development of competencies in ecological and mediational facets, ensure solid knowledge of mathematical content, employ adequate pedagogical strategies, and understand the relationship between the environment and mathematics. These findings offer insights into the prevailing trend of integrating ESD into mathematics education through ESD elements and didactic competencies, as evidenced by recent studies involving both practicing and pre-service teachers. This understanding may promote the enhancement of the quality of mathematics education practices within this domain.

Large numbers of cells are generally required for quantitative global proteome profiling due to surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) method termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-β-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for ‘all-in-one’ one-pot sample preparation. This ‘all-in-one’ method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics., Tsai, Zhang, Scholten et al. develop a surfactant- assisted one-pot sample preparation coupled with mass spectrometry method (SOP-MS) for label-free global single-cell proteomics. This method allows researchers to measure hundreds of proteins from single human cells, suggesting its utility for quantitative single-cell proteomics.

Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We developed a novel tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3,000 (>9,500) phosphopeptides from 1 (10) µg of cell lysate using the label-free method without a spectral library. It also enabled precise quantification of ∼600 phosphopeptides from 100 cells sorted by FACS (single-cell level input for the enriched phosphopeptides) and ∼700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 µm (equivalent to ∼100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.

CDK8 is a key subunit of Mediator complex, a large multiprotein complex that is a fundamental part of the conserved eukaryotic transcriptional machinery. However, the biological functions of CDK8 in plant abiotic stress responses remain largely unexplored. Here, we demonstrated CDK8 as a critical regulator in the abscisic acid (ABA) signaling and drought response pathways in Arabidopsis. Compared to wild-type, cdk8 mutants showed reduced sensitivity to ABA, impaired stomatal apertures and hypersensitivity to drought stress. Transcriptomic and chromatin immunoprecipitation analysis revealed that CDK8 positively regulates the transcription of several ABA-responsive genes, probably through promoting the recruitment of RNA polymerase II to their promoters. We discovered that both CDK8 and SnRK2.6 interact physically with an ERF/AP2 transcription factor RAP2.6, which can directly bind to the promoters of RD29A and COLD-REGULATED 15A (COR15A) with GCC or DRE elements, thereby promoting their expression. Importantly, we also showed that CDK8 is essential for the ABA-induced expression of RAP2.6 and RAP2.6-mediated upregulation of ABA-responsive genes, indicating that CDK8 could link the SnRK2.6-mediated ABA signaling to RNA polymerase II to promote immediate transcriptional response to ABA and drought signals. Overall, our data provide new insights into the roles of CDK8 in modulating ABA signaling and drought responses.

In Arabidopsis, the plasma membrane transporter PUT3 is important to maintain the cellular homeostasis of polyamines and plays a role in stabilizing mRNAs of some heat-inducible genes. The plasma membrane Na+ /H+ transporter SOS1 and the protein kinase SOS2 are two salt-tolerance determinants crucial for maintaining intracellular Na+ and K+ homeostasis. Here, we report that PUT3 genetically and physically interacts with SOS1 and SOS2, and these interactions modulate PUT3 transport activity. Overexpression of PUT3 (PUT3OE) results in hypersensitivity of the transgenic plants to polyamine and paraquat. The hypersensitivity of PUT3OE is inhibited by the sos1 and sos2 mutations, which indicates that SOS1 and SOS2 are required for PUT3 transport activity. A protein interaction assay revealed that PUT3 physically interacts with SOS1 and SOS2 in yeast and plant cells. SOS2 phosphorylates PUT3 both in vitro and in vivo. SOS1 and SOS2 synergistically activate the polyamine transport activity of PUT3, and PUT3 also modulates SOS1 activity by activating SOS2 in yeast cells. Overall, our findings suggest that both plasma-membrane proteins PUT3 and SOS1 could form a complex with the protein kinase SOS2 in response to stress conditions and modulate the transport activity of each other through protein interactions and phosphorylation.

Protein kinases are major regulatory components in almost all cellular processes in eukaryotic cells. By adding phosphate groups, protein kinases regulate the activity, localization, protein–protein interactions, and other features of their target proteins. It is known that protein kinases are central components in plant responses to environmental stresses such as drought, high salinity, cold, and pathogen attack. However, only a few targets of these protein kinases have been identified. Moreover, how these protein kinases regulate downstream biological processes and mediate stress responses is still largely unknown. In this study, we introduce a strategy based on isotope-labeled in vitro phosphorylation reactions using in vivo phosphorylated peptides as substrate pools and apply this strategy to identify putative substrates of nine protein kinases that function in plant abiotic and biotic stress responses. As a result, we identified more than 5,000 putative target sites of osmotic stress-activated SnRK2.4 and SnRK2.6, abscisic acid-activated protein kinases SnRK2.6 and casein kinase 1-like 2 (CKL2), elicitor-activated protein kinase CDPK11 and MPK6, cold-activated protein kinase MPK6, H 2 O 2 -activated protein kinase OXI1 and MPK6, and salt-induced protein kinase SOS1 and MPK6, as well as the low-potassium-activated protein kinase CIPK23. These results provide comprehensive information on the role of these protein kinases in the control of cellular activities and could be a valuable resource for further studies on the mechanisms underlying plant responses to environmental stresses.

Osmoregulation is important for plant growth, development and response to environmental changes. SNF1-related protein kinase 2s (SnRK2s) are quickly activated by osmotic stress and are central components in osmotic stress and abscisic acid (ABA) signaling pathways; however, the upstream components required for SnRK2 activation and early osmotic stress signaling are still unknown. Here, we report a critical role for B2, B3 and B4 subfamilies of Raf-like kinases (RAFs) in early osmotic stress as well as ABA signaling in Arabidopsis thaliana. B2, B3 and B4 RAFs are quickly activated by osmotic stress and are required for phosphorylation and activation of SnRK2s. Analyses of high-order mutants of RAFs reveal critical roles of the RAFs in osmotic stress tolerance and ABA responses as well as in growth and development. Our findings uncover a kinase cascade mediating osmoregulation in higher plants., Rapid activation of SnRK2 kinases is central to plant responses to osmotic stress and abscisic acid. Here the authors show that a group of Raf-like kinases are very quickly activated by osmotic stress, and then phosphorylate and activate SnRK2s.

Clade A PP2C phosphatases are central components of the ABA-receptor coupled core signaling pathway, and are involved in multiple stress responses and developmental processes. However, the direct targets or partner proteins of A clade PP2Cs that participate in these biological processes are largely unknown. Here, we used a TurboID-based proximity labeling method to identify putative associated proteins of one A clade PP2C phosphatase, ABI1. By combining the results from affinity purification or proximity labeling of biotinylated proteins, we identified more than four hundred putative ABI1-associated proteins, including dozens of known ABI1-intreacting proteins, as well as proteins involved in TOR signaling, phospho-regulation, and other biological processes. We found that RAFs, a group of protein kinases that phosphorylate and activate SnRK2s in ABA and osmotic stress signaling, are direct substrates of ABI1. A conserved serine residue located in the P-loop of the kinase domain, corresponding to Ser619 in RAF3, is a major functional ABI1 target site. ABI1-mediated dephosphorylation on this site strongly promotes the kinase activity of most B2 and B3 RAFs. Thus, ABI1 has dual functions in ABA signaling by dephosphorylating and inhibiting SnRK2 to prevent SnRK2 activation in unstressed conditions, while dephosphorylating B2 and B3 subgroup RAFs to maintain their basal kinase activity. PP2C-mediated dephosphorylation at the conserved serine residue may be a mechanism for RAF activation in both plants and animals, with potential implications for tumorigenesis in humans.

Mass spectrometry (MS)-based phosphoproteomics is a powerful tool for investigating cell signaling, yet it remains challenging to study plant phosphoproteomes due to the low yield of cell lysis and high complexity of plant lysate. Here we report a streamlined sample preparation workflow to analyze plant phosphoproteomes in a high-throughput manner. This workflow addresses the problem of low yield in the lysis step and eliminates the interferences of pigments and metabolites in plant lysate. Integrating chemical labeling and high pH reverse phase fractionation with this workflow achieves in-depth phosphoproteomic coverage. Notably, the scalability of this approach is demonstrated by systematically analyzing the effect of long-term cold stress in the perturbation of the tomato phosphoproteome. Identification of more than 30,000 phosphopeptides from tomato leaves and more than 5000 kinase-substrate pairs from Arabidopsis create the largest phosphoproteomic and signaling network resource to date.

Multicellular organisms such as plants contain different types of cells with specialized functions. Analyzing the characteristics of each type of cells will reveal specific cell functions and enhance understanding of how an organism organizes and works. Due to the lack of a method to enrich the particular type of cells and the limited sensitivity and capability of mass spectrometry-based technologies, comprehensive analyses of the proteome in a single plant cell are still impossible. Here we report a high-sensitive and efficient cell-type-specific proteomic pipeline, combining simplified flow cytometry-based fluorescent cell-sorting for fluorescent protoplasts and an optimized nano-scale proteomics method, which allows us to identify more than 1,000 unique proteins from 500 guard cell protoplasts. By this method, we performed an in-depth, quantitative comparison between the proteomics of guard cells and mesophyll cells, and revealed that the enrichment of signal transduction-related proteins enables guard cells to respond to various environmental stimuli quickly. This method was also applied to profile the proteome of different cells in root tips of Arabidopsis and rice seedlings. Thus, this pipeline is applicable to various types of cells in plant or non-plant systems to acquire systemic knowledge of how cells work specifically and in highly organized multiple cell organisms.

Brassinosteroid (BR) regulates a wide range of physiological responses through the activation of BRASSINAZOLE RESISTANT1 (BZR1), whose activity is tightly controlled by its phosphorylation status and degradation. Although BZR1 appears to be degraded in distinct ways in response to different hormonal or environmental cues, little is known about how BR signaling regulates its degradation. Here we show that the BR-regulated U-box protein PUB40 mediates the proteasomal degradation of BZR1 in a root-specific manner in Arabidopsis (Arabidopsis thaliana). BZR1 levels were strongly reduced by plant U-box40 (PUB40) overexpression, whereas the pub39 pub40 pub41 mutant accumulated much more BZR1 than wild type in roots. The bzr1-1D gain-of-function mutation reduced the interaction with PUB40, which suppressed PUB40-mediated BZR1 degradation in roots. The cell layer-specific expression of PUB40 in roots helps induce selective BZR1 accumulation in the epidermal layer. Both BR treatment and loss-of-function of PUB40 expanded BZR1 accumulation to most cell layers. In addition, BZR1 accumulation increased the resistance of pub39 pub40 pub41 to low inorganic phosphate availability, as observed in bzr1-1D BRASSINOSTEROID-INSENSITIVE2-induced phosphorylation of PUB40, which mainly occurs in roots, gives rise to BZR1 degradation through enhanced binding of PUB40 to BZR1 and PUB40's stability. Our results suggest a molecular mechanism of root-specific BZR1 degradation regulated by BR signaling.

Olfactory dysfunction is related to several clinical neurodegenerative diseases, such as Alzheimer’s disease, multiple sclerosis, degenerative ataxias, Parkinson’s disease, and so on. Owing to the individual difference in the sensory adaption of human smell function, the olfactory responses usually exhibit large inter-individual difference and change over time after repeated stimulations. The traditional analysis tools, such as statistical parametric mapping (SPM) in functional magnetic resonance imaging technique (fMRI) analysis, utilize the paradigm-based linear correlation and statistical techniques to discriminate the activation areas from background activities. However, these traditional approaches extract olfactory-induced responses using the stereotypic template or paradigm generated model. The olfactory-induced hemodynamic responses affected by internal/external events, such as changes of smell fatigue and attention, are not considered and therefore could result in misleading results. In this paper, owing to the stochastic characteristic of olfactory-induced responses, we adopted multivariate empirical mode decomposition (MEMD) to extract olfactory-induced hemodynamic responses in MRI blood-oxygen-level dependent (BOLD) signals. The MEMD is an efficient data-driven approach to extract nonlinear and non-stationary signals, which is an improved method to expand traditional empirical mode decomposition (EMD) from one channel to multi-channel processing. We applied MEMD to decompose time series of BOLD signals from each slice into multivariate intrinsic mode functions (IMF). The MEMD enables common features of different scales in an image slice to be arranged in distinct IMFs. Each IMF is an analytic, self-constructed, well-defined, and data-driven function with time-varying frequencies. Each IMF was examined by checking its correlation with the paradigm-generated template. The task-related IMFs were chosen to reconstruct olfactory-induced hemodynamic responses. The group analysis of MEMD-processed data showed olfactory-induced activations in the anterior cingulate cortex and middle frontal gyrus.

The objective of our study is to know the perception of Chilean university students toward the pedagogical methodologies in online English classes during the COVID-19 pandemic. We applied a questionnaire (built by fifteen experts, validated through a pilot test of 34 students, and improved with licensed 21.0 version SPSS in terms of consistency) to 341 students from different university programs. We analyzed the responses quantitatively and qualitatively and found out that 75% of the 341 students surveyed have shown agreement with the pedagogical methodologies applied in online classes and motivation in learning due to the permanent concern and communication of the teacher in charge. Besides, the students have also yielded satisfactory learning outcomes. These results reveal that the online English teaching of Chilean higher education during the pandemic has been good and indirectly show the willingness of Chilean university teachers to receive relevant and continuous training to deliver quality education. In 2022, many countries have returned to face-to-face classes, but we believe that the pedagogical methodologies used in this research could serve as a reference for teachers or researchers worldwide who wish to incorporate into their face-to-face classes a part of online teaching and do research on online English teaching.

Mass spectrometry (MS)-based phosphoproteomics is a powerful tool for investigating cell signaling, yet it remains challenging to study plant phosphoproteomes due to the low yield of cell lysis and high complexity of plant lysate. Here we report a streamlined sample preparation workflow to analyze plant phosphoproteomes in a high-throughput manner. This workflow addresses the problem of low yield in the lysis step and eliminates the interferences of pigments and metabolites in plant lysate. Integrating chemical labeling and high pH reverse phase fractionation with this workflow achieves in-depth phosphoproteomic coverage. Notably, the scalability of this approach is demonstrated by systematically analyzing the effect of long-term cold stress in the perturbation of the tomato phosphoproteome. Identification of more than 30,000 phosphopeptides from tomato leaves and more than 5000 kinase-substrate pairs from Arabidopsis create the largest phosphoproteomic and signaling network resource to date.