Transceptor NRT1.1 and receptor-kinase QSK1 complex controls PM H+-ATPase activity under low nitrate.

NRT1.1, a nitrate transceptor, plays an important role in nitrate binding, sensing, and nitrate-dependent lateral root (LR) morphology. However, little is known about NRT1.1-mediated nitrate signaling transduction through plasma membrane (PM)-localized proteins. Through in-depth phosphoproteome profiling using membranes of Arabidopsis roots, we identified receptor kinase QSK1 and plasma membrane H+-ATPase AHA2 as potential downstream components of NRT1.1 signaling in a mild low-nitrate (LN)-dependent manner. QSK1, as a functional kinase and molecular link, physically interacts with NRT1.1 and AHA2 at LN and specifically phosphorylates AHA2 at S899. Importantly, we found that LN, not high nitrate (HN), induces formation of the NRT1.1-QSK1-AHA2 complex in order to repress the proton efflux into the apoplast by increased phosphorylation of AHA2 at S899. Loss of either NRT1.1 or QSK1 thus results in a higher T947/S899 phosphorylation ratio on AHA2, leading to enhanced pump activity and longer LRs under LN. Our results uncover a regulatory mechanism in which NRT1.1, under LN conditions, promotes coreceptor QSK1 phosphorylation and enhances the NRT1.1-QSK1 complex formation to transduce LN sensing to the PM H+-ATPase AHA2, controlling the phosphorylation ratio of activating and inhibitory phosphorylation sites on AHA2. This then results in altered proton pump activity, apoplast acidification, and regulation of NRT1.1-mediated LR growth.

The LaCLE35 peptide modifies rootlet density and length in cluster roots of white lupin.

White lupin (lupinus albus L.) forms special bottlebrush-like root structures called cluster roots (CR) when phosphorus is low, to remobilise sparingly soluble phosphates in the soil. The molecular mechanisms that control the CR formation remain unknown. Root development in other plants is regulated by CLE  (CLAVATA3/ EMBRYO SURROUNDING REGION (ESR)-RELATED) peptides, which provide more precise control mechanisms than common phytohormones. This makes these peptides interesting candidates to be involved in CR formation, where fine tuning to environmental factors is required. In this study we present an analysis of CLE peptides in white lupin. The peptides LaCLE35 (RGVHy PSGANPLHN) and LaCLE55 (RRVHy PSCHy PDPLHN) reduced root growth and altered CR in hydroponically cultured white lupins. We demonstrate that rootlet density and rootlet length were locally, but not systemically, impaired by exogenously applied CLE35. The peptide was identified in the xylem sap. The inhibitory effect of CLE35 on root growth was attributed to arrested cell elongation in root tips. Taken together, CLE peptides affect both rootlet density and rootlet length, which are two critical factors for CR formation, and may be involved in fine tuning this peculiar root structure that is present in a few crops and many Proteaceae species, under low phosphorus availability.

Phosphoregulation in the N-terminus of NRT2.1 affects nitrate uptake by controlling the interaction of NRT2.1 with NAR2.1 and kinase HPCAL1 in Arabidopsis.

NRT2.1, the major high affinity nitrate transporter in roots, can be phosphorylated at five different sites within the N- and the C-terminus. Here, we characterized the functional relationship of two N-terminal phosphorylation sites, S21 and S28, in Arabidopsis. Based on a site-specific correlation network, we identified a receptor kinase (HPCAL1, AT5G49770), phosphorylating NRT2.1 at S21 and resulting in active nitrate uptake. HPCAL1 itself was regulated by phosphorylation at S839 and S870 within its kinase domain. In the active state, when S839 was dephosphorylated and S870 was phosphorylated, HPCAL1 was found to interact with the N-terminus of NRT2.1, mainly when S28 was dephosphorylated. Phosphorylation of NRT2.1 at S21 resulted in a reduced interaction of NRT2.1 with its activator NAR2.1, but nitrate transport activity remained. By contrast, phosphorylated NRT2.1 at S28 enhanced the interaction with NAR2.1, but reduced the interaction with HPCAL1. Here we identified HPCAL1 as the kinase affecting this phospho-switch through phosphorylation of NRT2.1 at S21.

Imaging of plant calcium-sensor kinase conformation monitors real time calcium-dependent decoding in planta.

Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here, we developed a genetically encoded Förster resonance energy transfer (FRET)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses.

Nuclear localization of HD-Zip IV transcription factor GLABRA2 is driven by Importin α.

GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor (TF) from Arabidopsis , is a developmental regulator of specialized cell types in the epidermis. GL2 contains a putative monopartite nuclear localization sequence (NLS) partially overlapping with its homeodomain (HD). We demonstrate that NLS deletion or alanine substitution of its basic residues (KRKRKK) affects nuclear localization and results in a loss-of-function phenotype. Fusion of the predicted NLS (GTNKRKRKKYHRH) to the fluorescent protein EYFP is sufficient for its nuclear localization in roots and trichomes. The functional NLS is evolutionarily conserved in a distinct subset of HD-Zip IV members including PROTODERMAL FACTOR2 (PDF2). Despite partial overlap of the NLS with the HD, genetic dissection of the NLS from PDF2 indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plant tissues followed by mass spectrometry-based proteomics identified Importin α (IMPα) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMPα-3 revealed major interacting residues. Split-ubiquitin cytosolic yeast two-hybrid assays suggest interaction between GL2 and four IMPα isoforms from Arabidopsis. Direct interactions were verified in vitro by co-immunoprecipitation with recombinant proteins. IMPα triple mutants ( impα- 1,2,3 ) exhibit defects in EYFP:GL2 nuclear localization in trichomes but not in roots, consistent with tissue-specific and redundant functions of IMPα isoforms in Arabidopsis . Taken together, our findings provide mechanistic evidence for IMPα-dependent nuclear localization of GL2 and other HD-Zip IV TFs in plants. One sentence summary: GLABRA2, a representative HD-Zip IV transcription factor from Arabidopsis , contains an evolutionarily conserved monopartite nuclear localization sequence that is recognized by Importin α for translocation to the nucleus, a process that is necessary for cell-type differentiation of the epidermis.

Lipid remodeling of contrasting maize (Zea mays L.) hybrids under repeated drought.

The role of recovery after drought has been proposed to play a more prominent role during the whole drought-adaption process than previously thought. Two maize hybrids with comparable growth but contrasting physiological responses were investigated using physiological, metabolic, and lipidomic tools to understand the plants' strategies of lipid remodeling in response to repeated drought stimuli. Profound differences in adaptation between hybrids were discovered during the recovery phase, which likely gave rise to different degrees of lipid adaptability to the subsequent drought event. These differences in adaptability are visible in galactolipid metabolism and fatty acid saturation patterns during recovery and may lead to a membrane dysregulation in the sensitive maize hybrid. Moreover, the more drought-tolerant hybrid displays more changes of metabolite and lipid abundance with a higher number of differences within individual lipids, despite a lower physiological response, while the responses in the sensitive hybrid are higher in magnitude but lower in significance on the level of individual lipids and metabolites. This study suggests that lipid remodeling during recovery plays a key role in the drought response of plants.

A high-confidence Physcomitrium patens plasmodesmata proteome by iterative scoring and validation reveals diversification of cell wall proteins during evolution.

Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large-scale systematic localization, defining a high-confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD-localized. Callose-degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium-like proteins, which also diversified into PD-localized and non-PD-localized members on distinct phylogenetic clades. Our high-confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large-scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle.

PEP7 acts as a peptide ligand for the receptor kinase SIRK1 to regulate aquaporin-mediated water influx and lateral root growth.

Plant receptors constitute a large protein family that regulates various aspects of development and responses to external cues. Functional characterization of this protein family and the identification of their ligands remain major challenges in plant biology. Previously, we identified plasma membrane-intrinsic sucrose-induced receptor kinase 1 (SIRK1) and Qian Shou kinase 1 (QSK1) as receptor/co-receptor pair involved in the regulation of aquaporins in response to osmotic conditions induced by sucrose. In this study, we identified a member of the elicitor peptide (PEP) family, namely PEP7, as the specific ligand of th receptor kinase SIRK1. PEP7 binds to the extracellular domain of SIRK1 with a binding constant of 1.44 ± 0.79 µM and is secreted to the apoplasm specifically in response to sucrose treatment. Stabilization of a signaling complex involving SIRK1, QSK1, and aquaporins as substrates is mediated by alterations in the external sucrose concentration or by PEP7 application. Moreover, the presence of PEP7 induces the phosphorylation of aquaporins in vivo and enhances water influx into protoplasts. Disturbed water influx, in turn, led to delayed lateral root development in the pep7 mutant. The loss-of-function mutant of SIRK1 is not responsive to external PEP7 treatment regarding kinase activity, aquaporin phosphorylation, water influx activity, and lateral root development. Taken together, our data indicate that the PEP7/SIRK1/QSK1 complex represents a crucial perception and response module that mediates sucrose-controlled water flux in plants and lateral root development.

The mycorrhiza-specific ammonium transporter ZmAMT3;1 mediates mycorrhiza-dependent nitrogen uptake in maize roots.

Most plant species can form symbioses with arbuscular mycorrhizal fungi (AMFs), which may enhance the host plant's acquisition of soil nutrients. In contrast to phosphorus nutrition, the molecular mechanism of mycorrhizal nitrogen (N) uptake remains largely unknown, and its physiological relevance is unclear. Here, we identified a gene encoding an AMF-inducible ammonium transporter, ZmAMT3;1, in maize (Zea mays) roots. ZmAMT3;1 was specifically expressed in arbuscule-containing cortical cells and the encoded protein was localized at the peri-arbuscular membrane. Functional analysis in yeast and Xenopus oocytes indicated that ZmAMT3;1 mediated high-affinity ammonium transport, with the substrate NH4+ being accessed, but likely translocating uncharged NH3. Phosphorylation of ZmAMT3;1 at the C-terminus suppressed transport activity. Using ZmAMT3;1-RNAi transgenic maize lines grown in compartmented pot experiments, we demonstrated that substantial quantities of N were transferred from AMF to plants, and 68%-74% of this capacity was conferred by ZmAMT3;1. Under field conditions, the ZmAMT3;1-dependent mycorrhizal N pathway contributed >30% of postsilking N uptake. Furthermore, AMFs downregulated ZmAMT1;1a and ZmAMT1;3 protein abundance and transport activities expressed in the root epidermis, suggesting a trade-off between mycorrhizal and direct root N-uptake pathways. Taken together, our results provide a comprehensive understanding of mycorrhiza-dependent N uptake in maize and present a promising approach to improve N-acquisition efficiency via plant-microbe interactions.

Regulatory Modules of Metabolites and Protein Phosphorylation in Arabidopsis Genotypes With Altered Sucrose Allocation.

Multi-omics data sets are increasingly being used for the interpretation of cellular processes in response to environmental cues. Especially, the posttranslational modification of proteins by phosphorylation is an important regulatory process affecting protein activity and/or localization, which, in turn, can have effects on metabolic processes and metabolite levels. Despite this importance, relationships between protein phosphorylation status and metabolite abundance remain largely underexplored. Here, we used a phosphoproteomics-metabolomics data set collected at the end of day and night in shoots and roots of Arabidopsis to propose regulatory relationships between protein phosphorylation and accumulation or allocation of metabolites. For this purpose, we introduced a novel, robust co-expression measure suited to the structure of our data sets, and we used this measure to construct metabolite-phosphopeptide networks. These networks were compared between wild type and plants with perturbations in key processes of sugar metabolism, namely, sucrose export (sweet11/12 mutant) and starch synthesis (pgm mutant). The phosphopeptide-metabolite network turned out to be highly sensitive to perturbations in sugar metabolism. Specifically, KING1, the regulatory subunit of SnRK1, was identified as a primary candidate connecting protein phosphorylation status with metabolism. We additionally identified strong changes in the fatty acid network of the sweet11/12 mutant, potentially resulting from a combination of fatty acid signaling and metabolic overflow reactions in response to high internal sucrose concentrations. Our results further suggest novel protein-metabolite relationships as candidates for future targeted research.

A Rapid and Universal Workflow for Label-Free-Quantitation-Based Proteomic and Phosphoproteomic Studies in Cereals.

Proteomics and phosphoproteomics are robust tools to analyze dynamics of post-transcriptional processes during growth and development. A variety of experimental methods and workflows have been published, but most of them were developed for model plants and have not been adapted to high-throughput platforms. Here, we describe an experimental workflow for proteome and phosphoproteome studies tailored to cereal crop tissues. The workflow consists of two parallel parts that are suitable for analyzing protein/phosphoprotein from total proteins and the microsomal membrane fraction. We present phosphoproteomic data regarding quantification coverage and analytical reproducibility for example preparations from maize root and shoot, wheat leaf, and a microsomal protein preparation from maize leaf. To enable users to adjust for tissue specific requirements, we provide two different methods of protein clean-up: traditional ethanol precipitation (PC) and a recently developed technology termed single-pot, solid-phase-enhanced sample preparation (SP3). Both the PC and SP3 methods are effective in the removal of unwanted substances in total protein crude extracts. In addition, two different methods of phosphopeptide enrichment are presented: a TiO2 -based method and Fe(III)-NTA cartridges on a robotized platform. Although the overall number of phosphopeptides is stable across protein clean-up and phosphopeptide enrichment methods, there are differences in the preferred phosphopeptides in each enrichment method. The preferred protocol depends on laboratory capabilities and research objective. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Total protein crude extraction Basic Protocol 2: Total protein clean-up with ethanol precipitation Alternate Protocol 1: Total protein clean-up with SP3 method Basic Protocol 3: Microsomal fraction protein extraction Basic Protocol 4: Protein concentration determination by Bradford assay Basic Protocol 5: In-solution digestion with trypsin Basic Protocol 6: Phosphopeptide enrichment with TiO2 Alternate Protocol 2: Phosphopeptide enrichment with Fe(III)-NTA cartridges Basic Protocol 7: Peptide desalting with C18 material Basic Protocol 8: LC-MS/MS analysis of (phospho)peptides and spectrum matching.

Combining analyses of metabolite profiles and phosphorus fractions to explore high phosphorus utilization efficiency in maize.

Phosphorus (P) limitation is a significant factor restricting crop production in agricultural systems, and enhancing the internal P utilization efficiency (PUE) of crops plays an important role in ensuring sustainable P use in agriculture. To better understand how P is remobilized to affect crop growth, we first screened P-efficient (B73 and GEMS50) and P-inefficient (Liao5114) maize genotypes at the same shoot P content, and then analyzed P pools and performed non-targeted metabolomic analyses to explore changes in cellular P fractions and metabolites in maize genotypes with contrasting PUE. We show that lipid P and nucleic acid P concentrations were significantly lower in lower leaves of P-efficient genotypes, and these P pools were remobilized to a major extent in P-efficient genotypes. Broad metabolic alterations were evident in leaves of P-efficient maize genotypes, particularly affecting products of phospholipid turnover and phosphorylated compounds, and the shikimate biosynthesis pathway. Taken together, our results suggest that P-efficient genotypes have a high capacity to remobilize lipid P and nucleic acid P and promote the shikimate pathway towards efficient P utilization in maize.

pH modulates interaction of 14-3-3 proteins with pollen plasma membrane H+ ATPases independently from phosphorylation.

Pollen grains transport the sperm cells through the style tissue via a fast-growing pollen tube to the ovaries where fertilization takes place. Pollen tube growth requires a precisely regulated network of cellular as well as molecular events including the activity of the plasma membrane H+ ATPase, which is known to be regulated by reversible protein phosphorylation and subsequent binding of 14-3-3 isoforms. Immunodetection of the phosphorylated penultimate threonine residue of the pollen plasma membrane H+ ATPase (LilHA1) of Lilium longiflorum pollen revealed a sudden increase in phosphorylation with the start of pollen tube growth. In addition to phosphorylation, pH modulated the binding of 14-3-3 isoforms to the regulatory domain of the H+ ATPase, whereas metabolic components had only small effects on 14-3-3 binding, as tested with in vitro assays using recombinant 14-3-3 isoforms and phosphomimicking substitutions of the threonine residue. Consequently, local H+ influxes and effluxes as well as pH gradients in the pollen tube tip are generated by localized regulation of the H+ ATPase activity rather than by heterogeneous localized distribution in the plasma membrane.

A systems-biology approach identifies co-expression modules in response to low phosphate supply in maize lines of different breeding history.

Plants cope with low phosphorus availability by adjusting growth and metabolism through transcriptomic and proteomic adaptations. We hypothesize that selected genotypes with distinct phosphorous (P) use efficiency covering the breeding history of European Flint heterotic pool provide a tool to reveal general and genotype-specific molecular responses to P limitation. We reconstructed protein and gene co-expression networks by weighted correlation network analysis and related these to phosphate deficiency-induced traits. In roots, low phosphate supply resulted in a decreasing abundance of proteins in the oxidative pentose phosphate pathway and a negative correlation with root and shoot phosphate content. We observed an increase in abundance and positive correlation with root and shoot phosphate content for proteins in sucrose biosynthesis, lipid metabolism, respiration and RNA processing. Purple acid phosphatases, superoxide dismutase and phenylalanine ammonia lyase were identified as being upregulated under low phosphate in all genotypes. Overall, correlations between protein and mRNA abundance changes were limited, with ribosomal proteins and the ubiquitin protein degradation pathway exclusively responding with protein abundance changes. Carbohydrate, phospho- and sulfo-lipid metabolism showed abundance changes at the protein and mRNA levels. These partially non-overlapping proteomic and transcriptomic adjustments to low phosphate suggest sugar and lipid metabolism as metabolic processes associated with improved P use efficiency specifically in Founder Flint lines. We identified a mitogen-activated protein kinase-kinase as a potential genotype-specific regulator of sucrose metabolism at low phosphate in Founder Flint line EP1. We conclude that, during breedingt of Elite Flint lines, regulation of primary metabolism has changed to result in a distinct low phosphate response in Founder lines.

Phosphorylation Site Motifs in Plant Protein Kinases and Their Substrates.

Protein phosphorylation is an important cellular regulatory mechanism affecting the activity, localization, conformation, and interaction of proteins. Protein phosphorylation is catalyzed by kinases, and thus kinases are the enzymes regulating cellular signaling cascades. In the model plant Arabidopsis, 940 genes encode for kinases. The substrate proteins of kinases are phosphorylated at defined sites, which consist of common patterns around the phosphorylation site, known as phosphorylation motifs. The discovery of kinase specificity with a preference of phosphorylation of certain motifs and application of such motifs in deducing signaling cascades helped to reveal underlying regulation mechanisms, and facilitated the prediction of kinase-target pairs. In this mini-review, we took advantage of retrieved data as examples to present the functions of kinase families along with their commonly found phosphorylation motifs from their substrates.

PhosPhAt 4.0: An Updated Arabidopsis Database for Searching Phosphorylation Sites and Kinase-Target Interactions.

The PhosPhAt 4.0 database contains information on Arabidopsis phosphorylation sites identified by mass spectrometry in large-scale experiments from different research groups. So far PhosPhAt 4.0 has been one of the most significant large-scale data resources for plant phosphorylation studies. Functionalities of the web application, besides display of phosphorylation sites, include phosphorylation site prediction and kinase-target relationships retrieval. Here, we present an overview and user instructions for the PhosPhAt 4.0 database, with strong emphasis on recent renewals regarding protein annotation by SUBA4.0 and Mapman4, and additional phosphorylation site information imported from other databases, such as UniProt. Here, we provide a user guide for the retrieval of phosphorylation motifs from the kinase-target database and how to visualize these results. The improvements incorporated into the PhosPhAt 4.0 database have produced much more functionality and user flexibility for phosphoproteomic analysis.

Comparison of path-based centrality measures in protein-protein interaction networks revealed proteins with phenotypic relevance during adaptation to changing nitrogen environments.

Plants must rapidly adapt to changes in nutrient conditions. Especially adaptations to changing nitrogen environments are very complex involving also major adjustments on the protein level. Here, we used a size-exclusion chromatography-coupled to mass spectrometry approach to study the dynamics of protein-protein interactions induced by transition from full nutrition to nitrogen starvation. Comparison of interaction networks established for each nutrient condition revealed a large overlap of proteins which were part of the protein-protein interaction network, but that same set of proteins underwent different interactions at each treatment. Network topology parameter betweenness centrality (BC) was found to best reflect the relevance of individual proteins in the information flow within each network. Changes in BC for individual proteins may therefore indicate their involvement in the cellular adjustments to the new condition. Based on this analysis, a set of proteins was identified showing high nitrogen-dependent changes in their BC values: The receptor kinase AT5G49770, co-receptor QSK1, and proton-ATPase AHA2. Mutants of those proteins showed a nitrate-dependent root growth phenotype. Individual interactions within the reconstructed network were tested using FRET-FLIM technology. Taken together, we present a systematic strategy comparing dynamic changes in protein-protein interaction networks based on their network parameters to identify regulatory nodes. SIGNIFICANCE: Protein-protein interactions are known to be important in cellular signaling events, but the dynamic changes in interaction networks induced by external stimuli are still rarely studied. We systematically analyzed how changes in the nutrient environment induced a rewiring of protein-protein interactions in roots. We observed small changes in overall protein abundances, but instead a rewiring of pairwise protein-protein interactions. Betweenness centrality was found to be the optimal network topology parameter to identify protein candidates with high relevance to the information flow in the (dynamic) network. Predicted interactions of those relevant nodes were confirmed in FLIM/FRET experiments and in phenotypic analysis. The network approach described here may be a useful application in dynamic network analysis more generally.

Plasma membrane calcineurin B-like calcium-ion sensor proteins function in regulating primary root growth and nitrate uptake by affecting global phosphorylation patterns and microdomain protein distribution.

The collective function of calcineurin B-like (CBL) calcium ion (Ca2+ ) sensors and CBL-interacting protein kinases (CIPKs) in decoding plasma-membrane-initiated Ca2+ signals to convey developmental and adaptive responses to fluctuating nitrate availability remained to be determined. Here, we generated a cbl-quintuple mutant in Arabidopsis thaliana devoid of these Ca2+ sensors at the plasma membrane and performed comparative phenotyping, nitrate flux determination, phosphoproteome analyses, and studies of membrane domain protein distribution in response to low and high nitrate availability. We observed that CBL proteins exert multifaceted regulation of primary and lateral root growth and nitrate fluxes. Accordingly, we found that loss of plasma membrane Ca2+ sensor function simultaneously affected protein phosphorylation of numerous membrane proteins, including several nitrate transporters, proton pumps, and aquaporins, as well as their distribution within plasma membrane microdomains, and identified a specific phosphorylation and domain distribution pattern during distinct phases of low and high nitrate responses. Collectively, these analyses reveal a central and coordinative function of CBL-CIPK-mediated signaling in conveying plant adaptation to fluctuating nitrate availability and identify a crucial role of Ca2+ signaling in regulating the composition and dynamics of plasma membrane microdomains.

Proteome profiling of repeated drought stress reveals genotype-specific responses and memory effects in maize.

Drought has become a major stress for agricultural productivity in temperate regions, such as central Europe. Thus, information on how crop plants respond to drought is important to develop tolerant hybrids and to ensure yield stability. Posttranscriptional regulation through changed protein abundances is an important mechanism of short-term response to stress events that has not yet been widely exploited in breeding strategies. Here, we investigated the response to repeated drought exposure of a tolerant and a sensitive maize hybrid in order to understand general protein abundance changes induced by singular drought or repeated drought events. In general, drought affected protein abundance of multiple pathways in the plant. We identified starch metabolism, aquaporin abundance, PSII proteins and histones as strongly associated with typical drought-induced phenotypes such as increased membrane leakage, osmolality or effects on stomatal conductance and assimilation rate. In addition, we found a strong effect of drought on nutrient assimilation, especially the sulfur metabolism. In general, pre-experience of mild drought before exposure to a more severe drought resulted in visible adaptations resulting in dampened phenotypes as well as lower magnitude of protein abundance changes.

Phosphoproteomic Analysis of Plant Membranes.

Mass spectrometry (MS) is a powerful tool to investigate plant phosphorylation dynamics on a system-wide scale (phosphoproteomics). Plant membrane phosphoproteomics enables elucidating regulatory patterns in membranes, such as kinase-target relationships in different signaling pathways. Here, we present "ShortPhos," an efficient and simple phosphoproteomics protocol for research on plant membrane proteins, which allows fast and efficient identification and quantification of phosphopeptides from small amounts of starting plant material and/or membrane proteins. This method improves upon the efficiency of plant membrane phosphoproteomics profiling and can be applied to the study of membrane-based signaling networks.