SPINDLY mediates O-fucosylation of hundreds of proteins and sugar-dependent growth in Arabidopsis.

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.

Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics.

Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.

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.

TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics.

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.

DIA-based phosphoproteomics identifies early phosphorylation events in response to EGTA and mannitol in Arabidopsis.

Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous research has demonstrated that osmotic stress rapidly induces calcium influx and signaling, along with the activation of a specific subset of protein kinases, notably the Raf-SnRK2 kinase cascades within minutes. However, the intricate interplay between calcium signaling and the activation of RAF-SnRK2 kinase cascades remains elusive. Here in this study, we discovered that Raf-like protein (RAF) kinases undergo hyperphosphorylation in response to osmotic shocks. Intriguingly, treatment with the calcium chelator EGTA robustly activates RAF-SnRK2 cascades, mirroring the effects of osmotic treatment. Utilizing high-throughput DIA-based phosphoproteomics, we unveiled the global impact of EGTA on protein phosphorylation. Beyond the activation of RAFs and sucrose non-fermenting-1-related protein kinase 2s (SnRK2s), EGTA treatment also activates mitogen-activated protein kinase (MAPKs) cascades, Calcium-dependent protein kinases (CDPKs), and receptor-like protein kinases, etc. Through overlapping assays, we identified potential roles of mitogen-activated protein kinase kinase kinase kinases (MAP4Ks) and receptor-like protein kinases in the osmotic-stress-induced activation of RAF-SnRK2 cascades. Our findings illuminate the regulation of phosphorylation and cellular events by Ca2+ signaling, offering insights into the (exocellular) Ca2+ deprivation during early hyperosmolality sensing and signaling.

Zebrafish Insights into Nanomaterial Toxicity: A Focused Exploration on Metallic, Metal Oxide, Semiconductor, and Mixed-Metal Nanoparticles.

Nanomaterials are widely used in various fields, and ongoing research is focused on developing safe and sustainable nanomaterials. Using zebrafish as a model organism for studying the potentially toxic effects of nanomaterials highlights the importance of developing safe and sustainable nanomaterials. Studies conducted on nanomaterials and their toxicity and potential risks to human and environmental health are vital in biomedical sciences. In the present review, we discuss the potential toxicity of nanomaterials (inorganic and organic) and exposure risks based on size, shape, and concentration. The review further explores various types of nanomaterials and their impacts on zebrafish at different levels, indicating that exposure to nanomaterials can lead to developmental defects, changes in gene expressions, and various toxicities. The review also covers the importance of considering natural organic matter and chorion membranes in standardized nanotoxicity testing. While some nanomaterials are biologically compatible, metal and semiconductor nanomaterials that enter the water environment can increase toxicity to aquatic creatures and can potentially accumulate in the human body. Further investigations are necessary to assess the safety of nanomaterials and their impacts on the environment and human health.

Suspension Trapping-Based Sample Preparation Workflow for In-Depth Plant Phosphoproteomics.

Plant phosphoproteomics provides a global view of phosphorylation-mediated signaling in plants; however, it demands high-throughput methods with sensitive detection and accurate quantification. Despite the widespread use of protein precipitation for removing contaminants and improving sample purity, it limits the sensitivity and throughput of plant phosphoproteomic analysis. The multiple handling steps involved in protein precipitation lead to sample loss and process variability. Herein, we developed an approach based on suspension trapping (S-Trap), termed tandem S-Trap-IMAC (immobilized metal ion affinity chromatography), by integrating an S-Trap micro-column with a Fe-IMAC tip. Compared with a precipitation-based workflow, the tandem S-Trap-IMAC method deepened the coverage of the Arabidopsis (Arabidopsis thaliana) phosphoproteome by more than 30%, with improved number of multiply phosphorylated peptides, quantification accuracy, and short sample processing time. We applied the tandem S-Trap-IMAC method for studying abscisic acid (ABA) signaling in Arabidopsis seedlings. We thus discovered that a significant proportion of the phosphopeptides induced by ABA are multiply phosphorylated peptides, indicating their importance in early ABA signaling and quantified several key phosphorylation sites on core ABA signaling components across four time points. Our results show that the optimized workflow aids high-throughput phosphoproteome profiling of low-input plant samples.

The methyl-CpG-binding protein MBD7 facilitates active DNA demethylation to limit DNA hyper-methylation and transcriptional gene silencing.

DNA methylation is a conserved epigenetic mark that plays important roles in plant and vertebrate development, genome stability, and gene regulation. Canonical Methyl-CpG-binding domain (MBD) proteins are important interpreters of DNA methylation that recognize methylated CG sites and recruit chromatin remodelers, histone deacetylases, and histone methyltransferases to repress transcription. Here, we show that Arabidopsis MBD7 and Increased DNA Methylation 3 (IDM3) are anti-silencing factors that prevent gene repression and DNA hypermethylation. MBD7 preferentially binds to highly methylated, CG-dense regions and physically associates with other anti-silencing factors, including the histone acetyltransferase IDM1 and the alpha-crystallin domain proteins IDM2 and IDM3. IDM1 and IDM2 were previously shown to facilitate active DNA demethylation by the 5-methylcytosine DNA glycosylase/lyase ROS1. Thus, MBD7 tethers the IDM proteins to methylated DNA, which enables the function of DNA demethylases that in turn limit DNA methylation and prevent transcriptional gene silencing.

Mapping proteome-wide targets of protein kinases in plant stress responses.

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, H2O2-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.

An Integrated Proteomic Strategy to Identify SHP2 Substrates.

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, in vivo global quantitative phosphoproteomics, and in vitro phosphatase 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.

The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalization.

It is well known that in addition to its classical role in protein turnover, ubiquitylation is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains: given that all ubiquitin units are identical, how do different elements of the sorting machinery recognize their specific cargoes? Our results indicate that the yeast Na+ pump Ena1 is an epsin (Ent1 and Ent2 in yeast)-specific cargo and that its internalization requires K1090, which likely undergoes Art3-dependent ubiquitylation. In addition, an Ena1 serine and threonine (ST)-rich patch, proposed to be targeted for phosphorylation by casein kinases, was also required for its uptake. Interestingly, our data suggest that this phosphorylation was not needed for cargo ubiquitylation. Furthermore, epsin-mediated internalization of Ena1 required a specific spatial organization of the ST patch with respect to K1090 within the cytoplasmic tail of the pump. We hypothesize that ubiquitylation and phosphorylation of Ena1 are required for epsin-mediated internalization.

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.

Plant U-Box40 Mediates Degradation of the Brassinosteroid-Responsive Transcription Factor BZR1 in Arabidopsis Roots.

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.

Leucine-rich repeat extensin proteins regulate plant salt tolerance in Arabidopsis.

The perception and relay of cell-wall signals are critical for plants to regulate growth and stress responses, but the underlying mechanisms are poorly understood. We found that the cell-wall leucine-rich repeat extensins (LRX) 3/4/5 are critical for plant salt tolerance in Arabidopsis The LRXs physically associate with the RAPID ALKALINIZATION FACTOR (RALF) peptides RALF22/23, which in turn interact with the plasma membrane-localized receptor-like protein kinase FERONIA (FER). The lrx345 triple mutant as well as fer mutant plants display retarded growth and salt hypersensitivity, which are mimicked by overexpression of RALF22/23 Salt stress promotes S1P protease-dependent release of mature RALF22 peptides. Treatment of roots with mature RALF22/23 peptides or salt stress causes the internalization of FER. Our results suggest that the LRXs, RALFs, and FER function as a module to transduce cell-wall signals to regulate plant growth and salt stress tolerance.

Quantitative Proteomics Reveals that GmENO2 Proteins Are Involved in Response to Phosphate Starvation in the Leaves of Glycine max L.

Soybean (Glycine max L.) is a major crop providing important source for protein and oil for human life. Low phosphate (LP) availability is a critical limiting factor affecting soybean production. Soybean plants develop a series of strategies to adapt to phosphate (Pi) limitation condition. However, the underlying molecular mechanisms responsible for LP stress response remain largely unknown. Here, we performed a label-free quantification (LFQ) analysis of soybean leaves grown under low and high phosphate conditions. We identified 267 induced and 440 reduced differential proteins from phosphate-starved leaves. Almost a quarter of the LP decreased proteins are involved in translation processes, while the LP increased proteins are accumulated in chlorophyll biosynthetic and carbon metabolic processes. Among these induced proteins, an enolase protein, GmENO2a was found to be mostly induced protein. On the transcriptional level, GmENO2a and GmENO2b, but not GmENO2c or GmENO2d, were dramatically induced by phosphate starvation. Among 14 enolase genes, only GmENO2a and GmENO2b genes contain the P1BS motif in their promoter regions. Furthermore, GmENO2b was specifically induced in the GmPHR31 overexpressing soybean plants. Our findings provide molecular insights into how soybean plants tune basic carbon metabolic pathway to adapt to Pi deprivation through the ENO2 enzymes.

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.

The plasma-membrane polyamine transporter PUT3 is regulated by the Na+ /H+ antiporter SOS1 and protein kinase SOS2.

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.

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.

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.

An Arabidopsis Nucleoporin NUP85 modulates plant responses to ABA and salt stress.

Several nucleoporins in the nuclear pore complex (NPC) have been reported to be involved in abiotic stress responses in plants. However, the molecular mechanism of how NPC regulates abiotic stress responses, especially the expression of stress responsive genes remains poorly understood. From a forward genetics screen using an abiotic stress-responsive luciferase reporter (RD29A-LUC) in the sickle-1 (sic-1) mutant background, we identified a suppressor caused by a mutation in NUCLEOPORIN 85 (NUP85), which exhibited reduced expression of RD29A-LUC in response to ABA and salt stress. Consistently, the ABA and salinity induced expression of several stress responsive genes such as RD29A, COR15A and COR47 was significantly compromised in nup85 mutants and other nucleoporin mutants such as nup160 and hos1. Subsequently, Immunoprecipitation and mass spectrometry analysis revealed that NUP85 is potentially associated with HOS1 and other nucleoporins within the nup107-160 complex, along with several mediator subunits. We further showed that there is a direct physical interaction between MED18 and NUP85. Similar to NUP85 mutations, MED18 mutation was also found to attenuate expression of stress responsive genes. Taken together, we not only revealed the involvement of NUP85 and other nucleoporins in regulating ABA and salt stress responses, but also uncovered a potential relation between NPC and mediator complex in modulating the gene expression in plants.