Reciprocal Regulation of the TOR Kinase and ABA Receptor Balances Plant Growth and Stress Response.

As sessile organisms, plants must adapt to variations in the environment. Environmental stress triggers various responses, including growth inhibition, mediated by the plant hormone abscisic acid (ABA). The mechanisms that integrate stress responses with growth are poorly understood. Here, we discovered that the Target of Rapamycin (TOR) kinase phosphorylates PYL ABA receptors at a conserved serine residue to prevent activation of the stress response in unstressed plants. This phosphorylation disrupts PYL association with ABA and with PP2C phosphatase effectors, leading to inactivation of SnRK2 kinases. Under stress, ABA-activated SnRK2s phosphorylate Raptor, a component of the TOR complex, triggering TOR complex dissociation and inhibition. Thus, TOR signaling represses ABA signaling and stress responses in unstressed conditions, whereas ABA signaling represses TOR signaling and growth during times of stress. Plants utilize this conserved phospho-regulatory feedback mechanism to optimize the balance of growth and stress responses.

A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Mediator kinase subunit CDK8 phosphorylates transcription factor TCP15 during tomato pollen development.

Pollen development in flowering plants has strong implications for reproductive success. Pollen DNA can be targeted to improve plant traits for yield and stress tolerance. In this study, we demonstrated that the Mediator subunit CYCLIN-DEPENDENT KINASE 8 (CDK8) is a key modulator of pollen development in tomato (Solanum lycopersicum). SlCDK8 knockout led to significant decreases in pollen viability, fruit yield, and fruit seed number. We also found that SlCDK8 directly interacts with transcription factor TEOSINTE BRANCHED1-CYCLOIDEA-PCF15 (SlTCP15) using yeast two-hybrid screens. We subsequently showed that SlCDK8 phosphorylates Ser 187 of SlTCP15 to promote SlTCP15 stability. Phosphorylated TCP15 directly bound to the TGGGCY sequence in the promoters of DYSFUNCTIONAL TAPETUM 1 (SlDYT1) and MYB DOMAIN PROTEIN 103 (SlMYB103), which are responsible for pollen development. Consistently, disruption of SlTCP15 resembled slcdk8 tomato mutants. In sum, our work identified a new substrate of Mediator CDK8 and revealed an important regulatory role of SlCDK8 in pollen development via cooperation with SlTCP15.

The tomato OST1-VOZ1 module regulates drought-mediated flowering.

Flowering is a critical agricultural trait that substantially affects tomato fruit yield. Although drought stress influences flowering time, the molecular mechanism underlying drought-regulated flowering in tomato remains elusive. In this study, we demonstrated that loss of function of tomato OPEN STOMATA 1 (SlOST1), a protein kinase essential for abscisic acid (ABA) signaling and abiotic stress responses, lowers the tolerance of tomato plants to drought stress. slost1 mutants also exhibited a late flowering phenotype under both normal and drought stress conditions. We also established that SlOST1 directly interacts with and phosphorylates the NAC (NAM, ATAF and CUC)-type transcription factor VASCULAR PLANT ONE-ZINC FINGER 1 (SlVOZ1), at residue serine 67, thereby enhancing its stability and nuclear translocation in an ABA-dependent manner. Moreover, we uncovered several SlVOZ1 binding motifs from DNA affinity purification sequencing analyses and revealed that SlVOZ1 can directly bind to the promoter of the major flowering-integrator gene SINGLE FLOWER TRUSS to promote tomato flowering transition in response to drought. Collectively, our data uncover the essential role of the SlOST1-SlVOZ1 module in regulating flowering in response to drought stress in tomato and offer insights into a novel strategy to balance drought stress response and flowering.

Loss of salt tolerance during tomato domestication conferred by variation in a Na+ /K+ transporter.

Domestication has resulted in reduced salt tolerance in tomato. To identify the genetic components causing this deficiency, we performed a genome-wide association study (GWAS) for root Na+ /K+ ratio in a population consisting of 369 tomato accessions with large natural variations. The most significant variations associated with root Na+ /K+ ratio were identified within the gene SlHAK20 encoding a member of the clade IV HAK/KUP/KT transporters. We further found that SlHAK20 transports Na+ and K+ and regulates Na+ and K+ homeostasis under salt stress conditions. A variation in the coding sequence of SlHAK20 was found to be the causative variant associated with Na+ /K+ ratio and confer salt tolerance in tomato. Knockout mutations in tomato SlHAK20 and the rice homologous genes resulted in hypersensitivity to salt stress. Together, our study uncovered a previously unknown molecular mechanism of salt tolerance responsible for the deficiency in salt tolerance in cultivated tomato varieties. Our findings provide critical information for molecular breeding to improve salt tolerance in tomato and other crops.

ZmELF6-ZmPRR37 module regulates maize flowering and salt response.

The control of flowering time in maize is crucial for reproductive success and yield, and it can be influenced by environmental stresses. Using the approaches of Ac/Ds transposon and transposable element amplicon sequencing techniques, we identified a Ds insertion mutant in the ZmPRR37 gene. The Ds insertion showed a significant correlation with days to anthesis. Further research indicated that ZmPRR37-CR knockout mutants exhibited early flowering, whereas ZmPRR37-overexpression lines displayed delayed flowering compared to WT under long-day (LD) conditions. We demonstrated that ZmPRR37 repressed the expression of ZmNF-YC2 and ZmNF-YA3 to delay flowering. Association analysis revealed a significant correlation between flowering time and a SNP2071-C/T located upstream of ZmPRR37. The SNP2071-C/T impacted the binding capacity of ZmELF6 to the promoter of ZmPRR37. ZmELF6 also acted as a flowering suppressor in maize under LD conditions. Notably, our study unveiled that ZmPRR37 can enhance salt stress tolerance in maize by directly regulating the expression of ABA-responsive gene ZmDhn1. ZmDhn1 negatively regulated maize salt stress resistance. In summary, our findings proposed a novel pathway for regulating photoperiodic flowering and responding to salt stress based on ZmPRR37 in maize, providing novel insights into the integration of abiotic stress signals into floral pathways.

ZmC2H2-149 negatively regulates drought tolerance by repressing ZmHSD1 in maize.

Drought is a major abiotic stress that limits maize production worldwide. Therefore, it is of great importance to improve drought tolerance in crop plants for sustainable agriculture. In this study, we examined the roles of Cys2 /His2 zinc-finger-proteins (C2H2-ZFPs) in maize's drought tolerance as C2H2-ZFPs have been implicated for plant stress tolerance. By subjecting 150 Ac/Ds mutant lines to drought stress, we successfully identified a Ds-insertion mutant, zmc2h2-149, which shows increased tolerance to drought stress. Overexpression of ZmC2H2-149 in maize led to a decrease in both drought tolerance and crop yield. DAP-Seq, RNA-Seq, Y1H and LUC assays additionally showed that ZmC2H2-149 directly suppresses the expression of a positive drought tolerance regulator, ZmHSD1 (hydroxysteroid dehydrogenase 1). Consistently, the zmhsd1 mutants exhibited decreased drought tolerance and grain yield under water deficit conditions compared to their respective wild-type plants. Our findings thus demonstrated that ZmC2H2-149 can regulate ZmHSD1 for drought stress tolerance in maize, offering valuable theoretical and genetic resources for maize breeding programmes that aim for improving drought tolerance.

A natural promoter variation of SlBBX31 confers enhanced cold tolerance during tomato domestication.

Cold stress affects crop growth and productivity worldwide. Understanding the genetic basis of cold tolerance in germplasms is critical for crop improvement. Plants can coordinate environmental stimuli of light and temperature to regulate cold tolerance. However, it remains unknown which gene in germplasms could have such function. Here, we utilized genome-wide association study (GWAS) to investigate the cold tolerance of wild and cultivated tomato accessions and discovered that increased cold tolerance is accompanied with tomato domestication. We further identified a 27-bp InDel in the promoter of the CONSTANS-like transcription factor (TF) SlBBX31 is significantly linked with cold tolerance. Coincidentally, a key regulator of light signalling, SlHY5, can directly bind to the SlBBX31 promoter to activate SlBBX31 transcription while the 27-bp InDel can prevent S1HY5 from transactivating SlBBX31. Parallel to these findings, we observed that the loss of function of SlBBX31 results in impaired tomato cold tolerance. SlBBX31 can also modulate the cold-induced expression of several ERF TFs including CBF2 and DREBs. Therefore, our study has uncovered that SlBBX31 is possibly selected during tomato domestication for cold tolerance regulation, providing valuable insights for the development of hardy tomato varieties.

Genome-wide association study of eigenvectors provides genetic insights into selective breeding for tomato metabolites.

BACKGROUND: Long-term domestication and intensive breeding of crop plants aim to establish traits desirable for human needs, and characteristics related to yield, disease resistance, and postharvest storage have traditionally received considerable attention. These processes have led also to negative consequences, as is the case of loss of variants controlling fruit quality, for instance in tomato. Tomato fruit quality is directly associated to metabolite content profiles; however, a full understanding of the genetics affecting metabolite content during tomato domestication and improvement has not been reached due to limitations of the single detection methods previously employed. Here, we aim to reach a broad understanding of changes in metabolite content using a genome-wide association study (GWAS) with eigenvector decomposition (EigenGWAS) on tomato accessions. RESULTS: An EigenGWAS was performed on 331 tomato accessions using the first eigenvector generated from the genomic data as a "phenotype" to understand the changes in fruit metabolite content during breeding. Two independent gene sets were identified that affected fruit metabolites during domestication and improvement in consumer-preferred tomatoes. Furthermore, 57 candidate genes related to polyphenol and polyamine biosynthesis were discovered, and a major candidate gene chlorogenate: glucarate caffeoyltransferase (SlCGT) was identified, which affected the quality and diseases resistance of tomato fruit, revealing the domestication mechanism of polyphenols. CONCLUSIONS: We identified gene sets that contributed to consumer liking during domestication and improvement of tomato. Our study reports novel evidence of selective sweeps and key metabolites controlled by multiple genes, increasing our understanding of the mechanisms of metabolites variation during those processes. It also supports a polygenic selection model for the application of tomato breeding.

ZmERF21 directly regulates hormone signaling and stress-responsive gene expression to influence drought tolerance in maize seedlings.

Drought stress adversely impacts crop development and yield. Maize frequently encounters drought stress during its life cycle. Improvement of drought tolerance is a priority of maize breeding programs. Here, we identified a novel transcription factor encoding gene, APETALA2 (AP2)/Ethylene response factor (ERF), which is tightly associated with drought tolerance in maize seedlings. ZmERF21 is mainly expressed in the root and leaf and it can be highly induced by polyethylene glycol treatment. Genetic analysis showed that the zmerf21 mutant plants displayed a reduced drought tolerance phenotype, accompanied by phenotypical and physiological changes that are commonly observed in drought conditions. Overexpression of ZmERF21 in maize significantly increased the chlorophyll content and activities of antioxidant enzymes under drought conditions. RNA-Seq and DNA affinity purification sequencing analysis further revealed that ZmERF21 may directly regulate the expression of genes related to hormone (ethylene, abscisic acid) and Ca signaling as well as other stress-response genes through binding to the promoters of potential target genes. Our results thereby provided molecular evidence of ZmERF21 is involved in the drought stress response of maize.

Advances in mass spectrometry-based phosphoproteomics for elucidating abscisic acid signaling and plant responses to abiotic stress.

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.

Secretion of Phospholipase Dδ Functions as a Regulatory Mechanism in Plant Innate Immunity.

Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function in multiple signal transduction cascades; however, the mechanisms regulating PLDs in response to pathogens remain unclear. Here, we found that Arabidopsis (Arabidopsis thaliana) PLDδ accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria graminis f. sp hordei Using fluorescence recovery after photobleaching and single-molecule analysis, we observed higher PLDδ density in the plasma membrane after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy showed that the interaction between PLDδ and the microdomain marker AtREMORIN1.3 (AtREM1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further unveiled a trade-off between brefeldin A (BFA)-resistant and -sensitive pathways in secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. Analysis of phosphatidic acid (PA), hydrogen peroxide, and jasmonic acid (JA) levels and expression of related genes indicated that the relocalization of PLDδ is crucial for its activation to produce PA and initiate reactive oxygen species and JA signaling pathways. Together, our findings revealed that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering PA-mediated signaling in plant innate immunity.plantcell;31/12/3015/FX1F1fx1.

A high-continuity and annotated tomato reference genome.

BACKGROUND: Genetic and functional genomics studies require a high-quality genome assembly. Tomato (Solanum lycopersicum), an important horticultural crop, is an ideal model species for the study of fruit development. RESULTS: Here, we assembled an updated reference genome of S. lycopersicum cv. Heinz 1706 that was 799.09 Mb in length, containing 34,384 predicted protein-coding genes and 65.66% repetitive sequences. By comparing the genomes of S. lycopersicum and S. pimpinellifolium LA2093, we found a large number of genomic fragments probably associated with human selection, which may have had crucial roles in the domestication of tomato. We also used a recombinant inbred line (RIL) population to generate a high-density genetic map with high resolution and accuracy. Using these resources, we identified a number of candidate genes that were likely to be related to important agronomic traits in tomato. CONCLUSION: Our results offer opportunities for understanding the evolution of the tomato genome and will facilitate the study of genetic mechanisms in tomato biology.

Sterols regulate endocytic pathways during flg22-induced defense responses in Arabidopsis.

The plant transmembrane receptor kinase FLAGELLIN SENSING 2 (FLS2) is crucial for innate immunity. Although previous studies have reported FLS2-mediated signal transduction and endocytosis via the clathrin-mediated pathway, whether additional endocytic pathways affect FLS2-mediated defense responses remains unclear. Here, we show that the Arabidopsis thaliana sterol-deficient mutant steroid methyltransferase 1 displays defects in immune responses induced by the flagellin-derived peptide flg22. Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) coupled with single-particle tracking showed that the spatiotemporal dynamics of FLS2-GFP changed on a millisecond time scale and that the FLS2-GFP dwell time at the plasma membrane increased in cells treated with a sterol-extracting reagent when compared with untreated counterparts. We further demonstrate that flg22-induced FLS2 clustering and endocytosis involves the sterol-associated endocytic pathway, which is distinct from the clathrin-mediated pathway. Moreover, flg22 enhanced the colocalization of FLS2-GFP with the membrane microdomain marker Flot 1-mCherry and FLS2 endocytosis via the sterol-associated pathway. This indicates that plants may respond to pathogen attacks by regulating two different endocytic pathways. Taken together, our results suggest the key role of sterol homeostasis in flg22-induced plant defense responses.

The transcription factor TaLAX1 interacts with Q to antagonistically regulate grain threshability and spike morphogenesis in bread wheat.

The domestication gene Q is largely responsible for the widespread cultivation of wheat because it confers multiple domestication traits. However, the underlying molecular mechanisms of how Q regulates these domestication traits remain unclear. In this study, we identify a Q-interacting protein TaLAX1, a basic helix-loop-helix transcription factor, through yeast two-hybrid assays. Using biochemical and genetic approaches, we explore the roles of TaLAX1 in regulating wheat domestication traits. Overexpression of TaLAX1 produces phenotypes, reminiscent of the q allele; loss-of-function Talax1 mutations confer compact spikes, largely similar to the Q-overexpression wheat lines. The two transcription factors TaLAX1 and Q disturb each other's activity to antagonistically regulate the expression of the lignin biosynthesis-related gene TaKNAT7-4D. More interestingly, a natural variation (InDel, +/- TATA), which occurs in the promoter of TaLAX1, is associated with the promoter activity difference between the D subgenome of bread wheat and its ancestor Aegilops tauschii accession T093. This study reveals that the transcription factor TaLAX1 physically interacts with Q to antagonistically regulate wheat domestication traits and a natural variation (InDel, +/- TATA) is associated with the diversification of TaLAX1 promoter activity.

Mediator tail module subunits MED16 and MED25 differentially regulate abscisic acid signaling in Arabidopsis.

MED25 has been implicated as a negative regulator of the abscisic acid (ABA) signaling pathway. However, it is unclear whether other Mediator subunits could associate with MED25 to participate in the ABA response. Here, we used affinity purification followed by mass spectrometry to uncover Mediator subunits that associate with MED25 in transgenic plants. We found that at least 26 Mediator subunits, belonging to the head, middle, tail, and CDK8 kinase modules, were co-purified with MED25 in vivo. Interestingly, the tail module subunit MED16 was identified to associate with MED25 under both mock and ABA treatments. We further showed that the disruption of MED16 led to reduced ABA sensitivity compared to the wild type. Transcriptomic analysis revealed that the expression of several ABA-responsive genes was significantly lower in med16 than those in wild type. Furthermore, we discovered that MED16 may possibly compete with MED25 to interact with the key transcription factor ABA INSENSITIVE 5 (ABI5) to positively regulate ABA signaling. Consistently, med16 and med25 mutants displayed opposite phenotypes in ABA response, cuticle permeability, and differential ABI5-mediated EM1 and EM6 expression. Together, our data indicate that MED16 and MED25 differentially regulate ABA signaling by antagonistically affecting ABI5-mediated transcription in Arabidopsis.

CDK8 is associated with RAP2.6 and SnRK2.6 and positively modulates abscisic acid signaling and drought response in Arabidopsis.

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.

Functions and regulatory framework of ZmNST3 in maize under lodging and drought stress.

The growth and development of maize are negatively affected by various abiotic stresses including drought, high salinity, extreme temperature, and strong wind. Therefore, it is important to understand the molecular mechanisms underlying abiotic stress resistance in maize. In the present work, we identified that a novel NAC transcriptional factor, ZmNST3, enhances maize lodging resistance and drought stress tolerance. ChIP-Seq and expression of target genes analysis showed that ZmNST3 could directly regulate the expression of genes related to cell wall biosynthesis which could subsequently enhance lodging resistance. Furthermore, we also demonstrated that ZmNST3 affected the expression of genes related to the synthesis of antioxidant enzyme secondary metabolites that could enhance drought resistance. More importantly, we are the first to report that ZmNST3 directly binds to the promoters of CESA5 and Dynamin-Related Proteins2A (DRP2A) and activates the expression of genes related to secondary cell wall cellulose biosynthesis. Additionally, we revealed that ZmNST3 directly binds to the promoters of GST/GlnRS and activates genes which could enhance the production of antioxidant enzymes in vivo. Overall, our work contributes to a comprehensive understanding of the regulatory network of ZmNST3 in regulating maize lodging and drought stress resistance.

Structurally ordered Pt3Co for oxygen reduction reaction prepared using polyvinylpyrrolidone as auxiliary dispersant.

Structurally ordered Pt3Co/C nanoparticles (NPs) were obtained via a spray paint drying method with an annealing treatment. The addition of a suitable dose of polyvinylpyrrolidone resulted in a narrow size distribution of the Pt3Co/C-600-1 NPs, an average particle size of ca. 4.6 nm, which may be due to the enhanced dispersion in aqueous solution resulting from the carbon support. The sample denoted as Pt3Co/C-600-1 NPs performs high activity for oxygen reduction reaction with the mass activity (MA) ca. 3 times higher than that of a commercial Pt/C catalyst at 0.9 V. Accelerated durability tests (ADTs) showed that Pt3Co/C-600-1 NPs exhibit superior stability with a minimal loss of 17.5% in MA at 0.9 V after 5000 cycles, while Pt/C catalysts show loss of 44.4%. This simple two-step strategy provides an effective way to prepare Pt-based catalysts for industrial application.

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

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.